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Platelet membrane glycoproteins implicated in ristocetin induced aggregation. Studies of the proteins on platelets from patients with Bernard Soulier syndrome and Von Willebrand's disease
Abstract
The antibiotic ristocetin only aggregates platelets in the presence of plasma von Willebrand factor. Platelets from patients with Bernard-Soulier syndrome do not aggregate upon addition of ristocetin although, in contrast to von Willebrand's disease, plasma levels of factor VIII complex (factor VIII clotting activity, von Willebrand factor activity, and von Willebrand antigen) are normal. The membrane surface of normal platelets was modified and compared to the surface of platelets from a patient with Bernard-Soulier syndrome in an attempt to identify the receptor involved in von Willebrand factor-ristocetin-induced aggregation. After the incubation of washed normal platelets with a preparation of ristocetin previously shown to contain a proteolytic contaminant, the aggregation response is significantly decreased on addition or normal plasma. Analaysis by gel electrophoresis of such platelets when stained for carbohydrate revealed a decrease in the relative amounts of membrane glycopro-eins. Chymotrypsin-treated normal platelets had less membrane glycoproteins in addition to giving a reduced aggregation response in ristocetin-induced aggregation. Staining of gels for protein and carbohydrate indicated that there was an extensive change in the surface of Bernard-Soulier platelets, whereas those from patients with von Willebrand's disease appeared the same as normal. Platelets from patients were labeled by the lactoperoxidase iodination technique. Not only was the relative intensity of staining of platelet-specific proteins and glycoproteins changed in Bernard-Soulier platelets, but the iodination of the glycoproteins on the membrane surface relative to other membrane constituents was lower. In contrast, platelets from patients with von Willebrand's disease showed a normal exposure of membrane components. These data suggest therefore that membrane glycoproteins may play a functional role in ristocetin-induced aggregation.
... Naim et al. observed that PNA, SBA, and RCA induced platelet agglutination; however, when platelets were challenged with vWF and exposed to PNA, agglutination was inhibited 41 . Nurden and Caen 42 , Jenkins et al. 43 , Cooper et al. 44 previously described gpI as a platelet receptor for vWF, which was also reinforced by Naim et al.. Kawakami and Hirano employed seven lectins (Con A, RCA, WGA, ONA, SBA, DBA, and UEA-1) to describe glycan distribution in human platelet membranes and observed differential affinities to the plasma membrane and open canalicular system (OCS), with Con A, PNA, RCA, and WGA presenting the highest affinity to the plasma membrane, the two former ones being the most intense in the OCS 45 . Double staining was performed using one of these three lectins (PSA, LCA, and WGA) and vWF, and we could not observe vWF labeling when the antibody was applied together with the lectins as the first or second step in the immunofluorescence, in contrast to a positive vWF expression when analyzed separately. ...
Megakaryocytes are cells described as present since the yolk sac during embryonic and fetal development, and responsible for homeostasis control. Despite robust knowledge available about platelets and coagulation, especially concerning their central role in homeostasis and diseases, gaps are still found in megakaryocyte development. Some studies have pointed out differences between adult and fetal megakaryocytes what prompted us to characterize the lectin-based carbohydrate residues from megakaryocytes in the fetal livers of mice at different gestational ages, a s well as the glycan residues expressed on the surface of other liver structures such as capsules and vessels.
... Naim et al. observed that PNA, SBA, and RCA induced platelet agglutination; however, when platelets were challenged with vWF and exposed to PNA, agglutination was inhibited 41 . Nurden and Caen 42 , Jenkins et al. 43 , Cooper et al. 44 previously described gpI as a platelet receptor for vWF, which was also reinforced by Naim et al.. Kawakami and Hirano employed seven lectins (Con A, RCA, WGA, ONA, SBA, DBA, and UEA-1) to describe glycan distribution in human platelet membranes and observed differential affinities to the plasma membrane and open canalicular system (OCS), with Con A, PNA, RCA, and WGA presenting the highest affinity to the plasma membrane, the two former ones being the most intense in the OCS 45 . Double staining was performed using one of these three lectins (PSA, LCA, and WGA) and vWF, and we could not observe vWF labeling when the antibody was applied together with the lectins as the first or second step in the immunofluorescence, in contrast to a positive vWF expression when analyzed separately. ...
Megakaryocytes are cells described as present since the yolk sac during embryonic and fetal development, and responsible for homeostasis control. Despite robust knowledge available about platelets and coagulation, especially concerning their central role in homeostasis and diseases, gaps are still found in megakaryocyte development.
Some studies have pointed out differences between adult and fetal megakaryocytes what prompted us to characterize the lectin-based carbohydrate residues from megakaryocytes in the fetal livers of mice at different gestational ages, a s well as the glycan residues expressed on the surface of other liver structures such as capsules and vessels.
... 153 Plasma VWF and FVIII levels and activity are normal. 154 Clinical manifestations include tendency to excessive bleeding from injuries, purpura, epistaxis, gingival bleeding, menorrhagia, and, more rarely, GIB and hematuria. 155 BSS is a genetic disorder that affects males and females in equal number. ...
Gastrointestinal bleeding (GIB) is serious, intractable, and potentially life-threatening condition. There is considerable heterogeneity in GIB phenotypes among congenital bleeding disorders (CBDs), making GIB difficult to manage. Although GIB is rarely encountered in CBDs, its severity in some patients makes the need for a comprehensive and precise assessment of underlying factors and management approaches imperative. Initial evaluation of GIB begins with assessment of hematological status; GIB should be ruled out in patients with chronic anemia, and in presentations that include hematemesis, hematochezia, or melena. High-risk patients with recurrent GIB require urgent interventions such as replacement therapy for treatment of coagulation factor deficiency (CFD). However, the best management strategy for CFD-related bleeding remains controversial. While several investigations have identified CBDs as potential risk factors for GIB, research has focused on assessing the risks for individual factor deficiencies and other CBDs. This review highlights recent findings on the prevalence, management strategies, and alternative therapies of GIB related to CFDs, and platelet disorders.
... The reduced levels of coagulation factor in infants results in alteration of conventional tests such as the activated partial thromboplastin time (aPTT). Unfortunately, there is no standardization for aPTT because of a wide variability of reagents and analyzing systems from center to center implying different reference ranges (13). Other global tests may have a different level of sensitivity to detect abnormalities in hemostatic proteins related to age. ...
Pediatric mechanical circulatory support (MCS) is considered a strategy for heart failure management as a bridge to recovery and transplantation or as a destination therapy. The final outcome is significantly impacted by the number of complications that may occur during MCS. Children on ventricular assist devices (VADs) and extracorporeal membrane oxygenation (ECMO) are at high risk for bleeding and thrombotic complications that are managed through anticoagulation. The first detailed guideline in pediatric VADs (Edmonton Anticoagulation and Platelet Inhibition Protocol) was based on conventional antithrombotic drugs, such as unfractionated heparin (UFH) and warfarin. UFH is the first-line anticoagulant in pediatric MCS, although its profile is not considered optimal in pediatric setting. The broad variation in heparin doses among children is associated with frequent occurrence of cerebrovascular accidents, bleeding, and thrombocytopenia. Direct thrombin inhibitors (DTIs) have been utilized as alternative strategies to heparin. Since 2018, bivalirudin has become the chosen anticoagulant in the long-term therapy of patients undergoing MCS implantation, according to the most recent protocols shared in North America. This article provides a review of the non-traditional anticoagulation strategies utilized in pediatric MCS, focusing on pharmacodynamics, indications, doses, and monitoring aspects of bivalirudin. Moreover, it exposes the efforts and the collaborations among different specialized centers, which are committed to an ongoing learning in order to minimize major complications in this special pediatric population. Further prospective trials regarding DTIs in a pediatric MCS setting are necessary and in specific well-designed randomized control trials between UFH and bivalirudin. To conclude, based on the reported literature, the clinical use of the bivalirudin in pediatric MCS seems to be a value added in controlling and maybe reducing thromboembolic complications. Further research is necessary to confirm all the results provided by this literature review.
... Ristomycin (also called ristocetin) is a type III glycopeptide compound which was isolated from Amycolatopsis lurida in the 1950s and used clinically to treat Gram-positive infections in 1957 (17)(18)(19). Due to ristomycin's side effects of inducing aggregation of Von Willebrand factor-dependent platelets and causing profound thrombocytopenia in some patients (20), it was later developed as a diagnostic cofactor to detect the widespread hereditary genetic disorder von Willebrand disease (21,22). The ristomycin BGCs were recently reported in A. lurida NRRL2420, Amycolatopsis japonica MG417-CF17, and Amycolatopsis sp. ...
Amycolatopsis sp. TNS106 harbors a ristomycin biosynthetic gene cluster ( asr ) in its genome and produces ristomycin A. Deletion of the sole cluster-situated StrR family regulatory gene asrR abolished the ristomycin A production and the transcription of asr genes orfs5 - 39 . Ristomycin A fermentation titer in Amycolatopsis sp. TNS106 was dramatically improved by overexpression of asrR and a heterologous StrR family regulatory gene bbr from the balhimycin BGC utilizing strong promoters and multiple gene copies. Ristomycin A production was improved by approximately 60-fold, resulting in fermentation titer of 4.01 g/L in flask culture, in one of the engineered strains. Overexpression of AsrR and Bbr upregulated transcription of tested asr biosynthetic genes, indicating that these asr genes were positively regulated by AsrR and Bbr. However, only the promoter region of the asrR -operon and the intergenic region upstream of orf12 were bound by AsrR and Bbr in gel retardation assays, suggesting that AsrR and Bbr directly regulated the asrR -operon and probably orfs12 - 14 , but no other asr biosynthetic genes. Further assays with synthetic short probes showed that AsrR and Bbr specifically bound not only probes containing the canonical inverted repeats but also a probe with only one 7-bp element of the inverted repeats in its native context. AsrR and Bbr have an N-terminal ParB-like domain and a central winged helix-turn-helix DNA-binding domain. Site-directed mutations indicated that the N-terminal ParB-like domain was involved in activation of ristomycin A biosynthesis and did not affect the DNA-binding activity of AsrR and Bbr.
IMPORTANCE
This study showed that overexpression of either native StrR family regulator (AsrR) or heterologous StrR family regulator (Bbr) dramatically improved ristomycin A production through increasing the transcription of biosynthetic genes directly or indirectly. The conserved ParB-like domain of AsrR and Bbr was demonstrated to be involved in the regulation of asr BGC expression. These findings provide new insights into the mechanism of StrR family regulators in the regulation of glycopeptide antibiotics biosynthesis. Furthermore, the regulators overexpression plasmids constructed in this study could serve as valuable tools for future utilization in strain improvement and genome mining for new glycopeptide antibiotics. In addition, ristomycin A is a type III glycopeptide antibiotic clinically used as a diagnostic reagent due to its side effect. The overproduction strains engineered in this study are ideal material for industrial production of ristomycin A.
... Shear induces exposure of the A1 domain in VWF anchored to subendothelial collagen and GPIb-IX -V binds to the exposed A1 domain. The hemostatic importance of GPIb-IX-V was first recognized when it was discovered that platelets from patients with the bleeding disorder Bernard-Soulier Syndrome (BSS) are highly deficient in or entirely lacking the complex [7,8]. Interestingly, patients with BSS present with low platelet counts (thrombocytopenia), potentially speaking to GPIb-IX-V's role in modulating platelet lifespan and clearance. ...
Platelet adhesion to the site of vascular damage is a critical early step in hemostasis. The platelet glycoprotein (GP) Ib-IX-V plays a key role in this step via its interaction with immobilized von Willebrand Factor (VWF). In addition to its well-known role in adhesion, GPIb-IX-V is critical for platelets’ survival in circulation and plays an important role in the regulation of platelet clearance. Several mechanisms of platelet clearance work in concert to maintain a normal platelet count and ensure that circulating platelets are functionally viable via removal of senescent or activated platelets. Furthermore, dysregulation of platelet clearance underlies several bleeding disorders. GPIb-IX-V is central to many physiological mechanisms of platelet clearance including clearance via glycan receptors, clearance of VWF-platelet complexes, and fast clearance of transfused platelets. GPIb-IX-V dependent clearance also underlies thrombocytopenia in several bleeding disorders, including von Willebrand disease (VWD) and immune thrombocytopenia. This review will cover physiological and pathological mechanisms of platelet clearance, focusing on the role of GPIb-IX-V.
... It is characterized by prolonged bleeding time, thrombocytopenia, giant platelets, and markedly decreased platelet aggregation with ristocetin, but normal aggregation with other agents.*-' The primary functional abnormality in BSD results from decreased adhesiveness of these platelets to exposed s~bendothelium~~' as a result of their decreased ability to bind von Willebrand factor ( V W F ) .~.~ Studies of platelet membrane glycoproteins (GP) by Caen in 1975" andPhillips et al in 1976" demonstrated a deficiency of GPIb in BSD patients. Later studies have also demonstrated lack of or marked deficiency of GPIX.'2,'3 ...
The underlying molecular basis for Bernard-Soulier Disease (BSD) is currently unknown. Platelets from patients with this autosomal recessive bleeding disorder have multiple abnormalities, including a markedly reduced von Willebrand factor-dependent adhesiveness due to a deficiency of the platelet membrane glycoprotein (GP) Ib/IX complex. In the present studies, we have used an intragenic restriction fragment length polymorphism (RFLP) for Taq I in the GPIb alpha gene to study linkage between this gene and the inheritance of BSD in a family with two affected siblings. Whereas the proband was heterozygous, showing both the 0.7 and 4.0 kb bands of this polymorphism (A/B), her affected brother was homozygous for the 0.7 kb band (A/A). Accordingly, these siblings did not inherit the same pair of GPIb alpha alleles from their parents. Additionally, one child of the proband was A/A, while the second studied child was A/B, with neither showing any evidence of BSD. No construct of heterozygosity or homozygosity for GPIb alpha alleles in this family is consistent with a model in which one or more defective GPIb alpha alleles could produce BSD. RFLP analysis with BamHI or HindIII showed entirely normal patterns in the patients, indicating the absence of any gross deletion of the GPIb alpha gene. GPIb alpha mRNA from patient platelets was reverse transcribed and subsequently amplified by the polymerase chain reaction, demonstrating the presence of GPIb alpha transcript. Furthermore, trace amounts of GPIb could be shown on the surface of patient platelets. Based on these results, a defect in the GPIb alpha gene is unlikely to be the cause of BSD in this family.
... Kanama zamanı ve PFA-100 ölçümlerinde uzama görülür (5). Trombosit fonksiyon testlerinde kollajen, ADP, epinefrin ve trombine cevap normal, ristosetine cevap anormaldir (1)(2)6). Akım sitometrik olarak trombosit yüzeyinde GpIb/V/IX (CD42a,b,c) ekspresyonunun düşüklüğünün gösterilmesi tanıya yardımcıdır (7). ...
... 35 Although others also showed a GPI defect in BSS platelets, difficulties in separating the large platelets from leukocytes led to controversy about the specificity of the defect with leukocyte DNA and protease contaminations leading to secondary modifications of the BSS platelet protein profiles. 30,36 Doubts about the specificity of the GPIb defect were quickly removed by studies combining lactoperoxidase-catalyzed 125 I-labeling or 3 H-labeling with CIE or one-and two-dimensional SDS-PAGE procedures (►Fig. 5). ...
The search for the components of the platelet surface that mediate platelet adhesion and platelet aggregation began for earnest in the late 1960s when electron microscopy demonstrated the presence of a carbohydrate-rich, negatively charged outer coat that was called the "glycocalyx." Progressively, electrophoretic procedures were developed that identified the major membrane glycoproteins (GP) that constitute this layer. Studies on inherited disorders of platelets then permitted the designation of the major effectors of platelet function. This began with the discovery in Paris that platelets of patients with Glanzmann thrombasthenia, an inherited disorder of platelet aggregation, lacked two major GP. Subsequent studies established the role for the GPIIb-IIIa complex (now known as integrin αIIbβ3) in binding fibrinogen and other adhesive proteins on activated platelets and the formation of the protein bridges that join platelets together in the platelet aggregate. This was quickly followed by the observation that platelets of patients with the Bernard-Soulier syndrome, with macrothrombocytopenia and a distinct disorder of platelet adhesion, lacked the carbohydrate-rich, negatively charged, GPIb. It was shown that GPIb, through its interaction with von Willebrand factor, mediated platelet attachment to injured sites in the vessel wall. What follows is a personal reflection on the studies that were performed in the early pioneering days.
... Bernard-Soulier syndrome is a congenital platelet disorder characterized by giant platelets and thrombocytopenia. The platelets show reduced adherence to components of injured vessel walls (Weiss et al, 1974) and reduced or absent aggregation in response to the antibiotic ristocetin ( Jenkins et al, 1976). The abnormalities can be ascribed to deficiencies of the GPIb-IX receptor complex (Nurden & Caen, 1975;Clemetson et al, 1982). ...
In Bernard-Soulier syndrome, a hereditary bleeding disorder, the platelets are deficient in the glycoprotein (GP) Ib–IX–V complex, a major receptor for the von Willebrand factor. The components of the complex are encoded by separate genes. Patients with this syndrome have a variable expression level of the receptor protein on platelets depending on the specific genetic abnormality. We describe a patient with life-long bleeding symptoms, who is homozygous for a unique stop mutation, Trp 498→ Stop in the GPIbα gene, resulting in a truncated GPIbα polypeptide chain. In contrast to previously reported truncated forms of GPIbα, this form contains a portion of the transmembranous domain as well as the juxtamembranous cysteines engaged in a disulphide bond with GPIbβ. Flow cytometry with GPIbα antibodies demonstrated the presence of GPIb on the patient’s platelets, although in reduced amounts compared to normal controls. GPIX was similarly detectable. Immunoblotting demonstrated that the patient synthesized a truncated GPIbα of the expected size of 130 K, which was, however, sensitive to proteolysis. These studies show that GPIbα lacking the intracytoplasmic tail can be expressed at the platelet surface provided elements of the transmembranous domain are present.
... In turn, the adhesion of platelets to subendothelial matrix proteins depends upon red blood cell size and deformability, the presence of divalent calcium ions, and specific multimeric vWF distribution (32)(33)(34)(35). Patients with Bernard-Soulier disease have platelets that do not bind to collagen under flow even in the presence of VIII/vWF, indicating that platelets from these patients lacked a vWF receptor (36), now identified as the 155 kDA glycoprotein platelet glycoprotein Ib (37)(38)(39)(40)(41). ...
The study of blood ex vivo can occur in closed or open systems, with or without flow. Microfluidic devices, which constrain fluids to a small (typically submillimeter) scale, facilitate analysis of platelet function, coagulation biology, cellular biorheology, adhesion dynamics, and pharmacology and, as a result, can be an invaluable tool for clinical diagnostics. An experimental session can accommodate hundreds to thousands of unique clotting, or thrombotic, events. Using microfluidics, thrombotic events can be studied on defined surfaces of biopolymers, matrix proteins, and tissue factor, under constant flow rate or constant pressure drop conditions. Distinct shear rates can be generated on a device using a single perfusion pump. Microfluidics facilitated both the determination of intraluminal thrombus permeability and the discovery that platelet contractility can be activated by a sudden decrease in flow. Microfluidic devices are ideal for multicolor imaging of platelets, fibrin, and phosphatidylserine and provide a human blood analog to mouse injury models. Overall, microfluidic advances offer many opportunities for research, drug testing under relevant hemodynamic conditions, and clinical diagnostics. Expected final online publication date for the Annual Review of Biomedical Engineering Volume 15 is July 11, 2013. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
... Fortunately, we were able to access two BSS patients for this work. Deficiencies in human GP1b, both genetic and acquired, are found in BSS [21], cardiopulmonary bypass (CPB) [22] surgery, and surgical trauma [23], and in targeted transgenic mice [24], and lead to bleeding diatheses, presumably due to defects in the binding of platelets to the subendothelium. Whether such a deficiency would alter rates of thrombus formation and ultimate strength of the thrombus in whole blood with exogenous soluble subendothelium components, was not known, and is one contribution from this work. ...
To delineate the critical features of platelets required for formation and stability of thrombi, thromboelastography and platelet aggregation measurements were employed on whole blood of normal patients and of those with Bernard-Soulier Syndrome (BSS) and Glanzmann's Thrombasthenia (GT). We found that separation of platelet activation, as assessed by platelet aggregation, from that needed to form viscoelastic stable whole blood thrombi, occurred. In normal human blood, ristocetin and collagen aggregated platelets, but did not induce strong viscoelastic thrombi. However, ADP, arachidonic acid, thrombin, and protease-activated-receptor-1 and -4 agonists, stimulated both processes. During this study, we identified the genetic basis of a very rare double heterozygous GP1b deficiency in a BSS patient, along with a new homozygous GP1b inactivating mutation in another BSS patient. In BSS whole blood, ADP responsiveness, as measured by thrombus strength, was diminished, while ADP-induced platelet aggregation was normal. Further, the platelets of 3 additional GT patients showed very weak whole blood platelet aggregation toward the above agonists and provided whole blood thrombi of very low viscoelastic strength. These results indicate that measurements of platelet counts and platelet aggregability do not necessarily correlate with generation of stable thrombi, a potentially significant feature in patient clinical outcomes.
Background:
Bernard Soulier Syndrome (BSS) is a rare autosomal recessive disorder due to deficiency or dysfunction of the glycoprotein GPIb-V-IX complex on the platelet surface. It is also known as hemorrhagiparous thrombocytic dystrophy or congenital hemorrhagiparous thrombocytic dystrophy. The patient usually presents with severe and prolonged bleeding along with characteristics of giant blood platelets and low platelet counts. Manifestations of BSS include epistaxis, gum bleeding, purpuric rashes, menorrhagia, rarely melena, and hematemesis. On the other hand, immune thrombocytopenic purpura (ITP) is an acquired autoimmune disorder in which there is accelerated platelet destruction and reduced platelet production. Isolated thrombocytopenia without fever, lymphadenopathy, and organomegaly usually lead to the diagnosis of immune thrombocytopenia.
Case presentation:
A 20 years old female presented with complaints of recurrent episodes of epistaxis since childhood and menorrhagia during menarche. She was misdiagnosed as ITP elsewhere. Later, based on thorough clinical examination and investigation, the diagnosis was confirmed as BSS.
Conclusion:
BSS should always be taken in the differential diagnosis of ITP, especially when persistent, refractory, and treated unsuccessfully with steroids or splenectomy.
Glycoprotein (GP) Ib-IX-V is the second most abundant platelet receptor for thrombin and other ligands crucial for hemostasis and thrombosis. Its activity is involved in platelet adhesion to vascular injury sites and thrombin-induced platelet aggregation. GPIb-IX-V is a heteromeric complex composed of four subunits, GPIbα, GPIbβ, GPV and GPIX, in a stoichiometric ratio that has been wildly debated. Despite its important physiological roles, the overall structure and molecular arrangement of GPIb-IX-V are not yet fully understood. Here, we purify stable and functional human GPIb-IX-V complex from reconstituted EXPi293F cells in high homogeneity, and perform biochemical and structural characterization of this complex. Single-particle cryo-electron microscopy structure of GPIb-IX-V is determined at ∼11 Å resolution, which unveils the architecture of GPIb-IX-V and its subunit organization. Size-exclusion chromatography-multi-angle static light scattering analysis reveals that GPIb-IX-V contains GPIb-IX and GPV at a 1:1 stoichiometric ratio and surface plasmon resonance assays show that association of GPV leads to slow kinetics of thrombin binding to GPIb-IX-V. Taken together, our results provide the first three-dimensional architecture of the intact GPIb-IX-V complex, which extends our understanding of the structure and functional mechanism of this complex in hemostasis and thrombosis.
Background:
Acquired von Willebrand syndrome (AVWS) is frequent in patients with myeloproliferative neoplasms (MPNs). For VWF functional evaluation, ristocetin cofactor activity by aggregometry (VWF: RCo) is considered the gold standard but have limitations, and automated activities measurement has been developed such as the HemosIl VWF:RCo Werfen® with particle agglutination (VWF:GPIbR).
Objectives:
To evaluate the performance of VWF:GPIbR with HemosIL VWF:RCo Werfen® (VWF:GPIbR) versus VWF:RCo in patients with thrombocytosis in the context of MPNs (T-MPNs) and in patients with secondary thrombocytosis (ST).
Patients/methods:
MPNs patients with thrombocytosis >450 G/L (T-MPNs) were compared to patients with secondary thrombocytosis (ST) due to inflammation or iron deficiency. VWF activity (VWF:Act) was analyzed using VWF:RCo or VWF:GPIbR. VWF analysis was completed by analysis of VWF multimers and VWF collagen binding (CB) assay (VWF:CB).
Results:
33 T-MPNs and 18 ST patients were included. Compared to aggregometry, evaluation of VWF:Act by VWF:GPIbR leaded to lower values in T-MPNs patients, but also in ST patients. Interestingly, whereas VWF:RCo/VWF:Ag ratio did not reveal differences between T-MPNs and ST patients, VWF:GPIbR/VWF:Ag ratio analysis allowed to suspect AVWS only in T-MPNs patients. Using the distribution of VWF multimers analysis and VWF:CB, we here demonstrated that VWF:GPIbR allows AVWS diagnosis in 9 T-MPNs as opposed to aggregometry.
Conclusions:
Evaluation of VWF:Act using VWF:GPIbR has a greater sensitivity compared to aggregometry to detect AVWS in T-MPNs patients.
The aim of this thesis was to study platelet function in whole blood, in an environment close to normal physiology. It was anticipated that such studies in whole blood would be more sensitive to in vivo changes than studies on isolated platelets, since all of the formed elements are present, there is less risk of in vitro activation, and labile substances should still be present. This approach was thought to be particularly applicable to platelet studies in blood from patients with prothrombotic states where the mechanism of thrombosis is unclear, and for examining anti-platelet drug efficacy. Existing methods were inadequate and it was first necessary to develop new techniques using electrical impedance. Sensitive and reproducible impedance methods were developed, and these were especially useful for blood samples with platelet counts below 50*10⁹/1. A particular type of aggregate was detected, requiringirreversible aggregation, thromboxane A2 generation, but not ADP secretion. Measurements in non-anticoagulated blood allowed a global view of haemostasis, reflecting interactions of platelets, neutrophils, thrombin, and fibrin. In the presence of calcium++, neutrophils and thrombin potentiated the aggregation response. There was excessive aggregation in non-anticoagulated blood from patients with multi-organ failure. This was related to increased neutrophil count, enhanced thrombin generation, and reciprocal cell activation. Such patients may benefit from eicosanoid antagonists and protease inhibitors to prevent unwarranted activation of coagulation and cellular defense mechanisms. Chronic arteriopaths showed reduced aggregation compatible with in vivo activation. Prostacyclin analogue infusions were less effective as judged ex vivo in whole blood compared to traditional techniques; continuous infusion resulted in progressively decreasing platelet sensitivity, rebound hyperaggregability, and increased serum thromboxane B2. Cigarette smoking caused increased platelet aggregation and adherence to vascular endothelium. Increased aggregation was seen in multi-organ failure, which traditional methods would not have detected. There appear to be multiple interactions between leucocytes and platelets.
Platelet activation results in two major changes at the platelet surface: a change in GPIIb- Illa, exposing the previously cryptic fibrinogen binding site and fusion of platelet and granule membranes, leading to the expression of the two known granule membrane proteins, GMP-140 (α-granule) and GP53 (lysosome). Anti-GPIIb-IIIa monoclonal antibodies (MAbs) were raised and characterised in functional and flow cytometric assays. Competitive binding studies, with these and a panel of anti-GPIIb-IIIa MAbs, identified distinct epitopes on the complex important in influencing fibrinogen receptor exposure. Platelet aggregation was totally inhibited by MAbs binding to two conformation-dependent sites, one on GPIIb-IIIa and one on GPIIIa. A second group of antibodies, heterogeneous in their inhibitory effects on platelet aggregation, bound to overlapping sites on GPIIb-IIIa, GPIIb and GPIIIa. Three conformation-specific sites were identified, which induced platelet aggregation in the absence of any other agonist. All inhibitory MAbs totally inhibited fibrinogen binding to ADP-stimulated platelets and evidence is presented that fibrinogen and von Willebrand Factor bind to GPIIb-IIIa in a comparable and competitive way. Anti-GMP-140and anti-GP53 MAbs, raised against activated platelets, were characterised in Western blotting and phenotypic studies. Anti-GP53 MAbs were heterogeneous in their reactivity with a number of cell types; in particular, one MAb, RFAC-4, failed to bind to platelets from albino patients: a finding that may reflect the homology of GP53 with the melanoma-associated antigen, ME491. Flow cytometric, antibody binding studies showed that whilst the expression of both GMP-140 and GP53 was up-regulated over very similar thrombin concentrations, their expression ADP-stimulated platelets differed: GMP-140 was not detected on these cells but a partial expression of GP53 was seen. Using a whole blood, flow cytometric method, which minimises platelet activation in vitro. GP53 was shown to be a useful marker of platelet activation in the ex vivo analysis of platelets in a several clinical conditions.
Platelet disorders in the adolescent female are comprised of congenital and acquired platelet disorders associated with a decrease in platelet number and/or abnormality in platelet function. Bleeding symptoms in patients with platelet disorders most commonly involve skin and mucous membrane bleeding. In females with platelet disorders, heavy menstrual bleeding is the most common bleeding symptom. In adolescents presenting for evaluation, a complete history of bleeding symptoms including menstrual history, history of excessive bleeding with dental and surgical procedures, family history of bleeding symptoms and diagnosed bleeding disorders, and medication history should be obtained. Bleeding assessment and screening tools may be useful. Available laboratory testing for platelet disorders in addition to platelet count includes light and electron microscopy, platelet aggregometry and secretion assays, flow cytometric analysis, and point-of-care tests. Molecular diagnostic testing can provide confirmatory testing. Management varies depending on the severity of the bleeding symptoms and may include antifibrinolytic agents, recombinant VIIa, DDAVP, and platelet transfusions. Hormonal agents may be required for management of heavy menstrual bleeding in adolescent females with platelet disorders. Thrombopoietin mimetics are useful in raising the platelet count in acute and chronic ITP and in congenital MYH9-related platelet disorders. Identification and management of iron deficiency anemia is important in adolescent females with platelet disorders.
The glycoprotein (GP)Ib‐IX receptor complex plays a critical role in platelet physiology and pathology. Its interaction with von Willebrand factor (VWF) on the subendothelial matrix instigates platelet arrest at the site of vascular injury, and is vital to primary hemostasis. Its reception to other ligands and counter‐receptors in the blood stream also contribute to various processes of platelet biology that are still being discovered. While its basic composition and its link to congenital bleeding disorders were well documented and firmly established more than 25 years ago, recent years have witnessed critical advances in the organization, dynamics, activation, regulation and functions of the GPIb‐IX complex. This review summarizes important findings and identifies questions that remain about this unique platelet mechanoreceptor complex.
Background:
Disruption of protein folding or inter-subunit interactions in the platelet glycoprotein (GP)Ib-IX complex leads to its abnormally low expression in the plasma membrane, the hallmark of Bernard-Soulier syndrome (BSS).
Objective:
To discover the molecular mechanism by which GPIbα in the absence of GPIbβ and GPIX subunits is targeted for rapid degradation.
Method:
The expression of GPIbα mutants with deletion or replacement of various domains were measured in transiently transfected Chinese hamster ovary (CHO) cells.
Results:
We report evidence to suggest that induction of the unfolded protein response by the unaccompanied mechanosensory domain (MSD) is a major factor for intracellular degradation and low expression of GPIbα. Removal of the MSD produced the first GPIbα variant that, even in the absence of GPIbβ and GPIX, expressed at a level comparable to that of wild-type GPIbα in the GPIb-IX complex, while retaining its native ligand-binding activity.
Conclusion:
Our finding has important implications on the molecular pathogenesis of BSS and the function of the GPIb-IX complex.
Platelet receptors are how platelets interact with their environment in order to function. They enable signals to pass to the platelet interior and change functional aspects of the platelet exterior. Major receptors (described in detail in other chapters) have well-established ligands and functions, but there are a large number of minor receptors whose roles and ligands are only starting to be identified. Some of the receptors are always present on the plasma membrane, but others are on granule membranes and are only exposed following exocytosis. While their major roles are in hemostasis, a growing number of “minor” receptors are involved in other platelet activities such as immunity or in regulating physiological functions. Some receptors can be downregulated by shape change or by induced proteolytic activity. Since platelets lack a nucleus, they are dependent on their receptors and exocytosis to react to new situations.
Platelets are small anucleate blood cells that are produced in the bone marrow from the cytoplasm of megakaryocytes. Circulating platelets are essential for primary hemostasis and also involved in pathological thrombosis. For the platelet hemostatic functions, platelet surface membrane glycoproteins are crucial to form platelet-subendothelial matrix and platelet-platelet interactions. At the site of blood vessel injury, platelets are captured by platelet GPIb-IX-V interaction with von Willebrand factor which bound to exposed collagen followed by direct platelet-collagen interaction by GPIa-IIa (integrin α2β1) and GPVI. Platelet fibrinogen receptor GPIIb-IIIa (integrin αIIbβ3) is the most abundant glycoprotein on platelet surface, and its affinity for fibrinogen is tightly regulated by inside-out signaling. The platelet-platelet interaction mediated by activated GPIIb-IIIa is necessary for platelet accumulation on the layer of adhered platelets at the injured vessel. Both quantitative and qualitative abnormalities in these platelet glycoproteins can be a cause of platelet dysfunctions and bleeding disorders. In addition, platelet glycoproteins are also important in the pathogenesis of idiopathic thrombocytopenic purpura (ITP). In the majority of patients with ITP, antiplatelet autoantibodies in plasma are directed against platelet glycoproteins especially GPIIb-IIIa and GPIb-IX-V.
Dysregulation of one or more elements in hemostatic system can cause an increased risk of bleeding or hypercoagulability (thrombosis). Highly complex platelets play an important role in hemostasis and thrombosis through adhesion to subendothelium, release of coagulation factors in granules, and aggregation. A hereditary or acquired deficiency in one or more procoagulant factors can lead to excessive bleeding. Hereditary or acquired deficiency or dysfunction of regulatory proteins such as antithrombin, protein C, and protein S can lead to a hypercoagulability. Polymorphisms such as factor V Leiden or prothrombin gene G20210A or rare mutations can cause hypercoagulability. Antiphospholipid syndrome or lupus anticoagulant is a common cause of acquired thrombophilia with increased risk of arterial and venous thrombosis. Many current diagnostic laboratory tests can be often affected by concurrent clinical conditions or anticoagulant therapy. To identify the patients in a risk of bleeding or hypercoagulability, a battery of diagnostic laboratory tests should be performed in a stepwise manner by diagnostic algorithm beginning with high-yield screening tests followed by appropriate specific confirmatory tests. Consultation and comprehensive narrative interpretation by coagulation specialists are of great importance for correct interpretation of the results related to clinical conditions.
Beim v. Willebrand-Jürgens-Syndrom (vWJS) handelt es sich um eine in den meisten Fällen hereditäre, autosomal dominant oder rezessiv vererbte, selten aber auch erworbene hämorrhagische Diathese mit einer quantitativen und/oder qualitativen Störung des als v. Willebrand-Faktor (VIII:vWF) bezeichneten großmolekularen Anteils des Faktor-VIII/v. Willebrand-Faktor-Komplexes.
Die Ursache für eine Reihe von mehr oder weniger ausgeprägten Blutungsneigungen ist in vielen Fällen der Mangel oder Veränderungen spezifischer Membranglycoproteine der Blutplättchen. Zwei seltene, ausgeprägte, kongenitale Blutungsübel, die Thrombasthenie Glanzmann und das Bernard-Soulier-Syndrom, bei denen der Glykoproteindefekt weitgehend aufgeklärt ist, haben der Erforschung der Membran-glykoproteine, ihrer Struktur, Funktion und Rolle bei der Blutstillung besonderen Impetus verliehen. Defekte in anderen Membranglykoproteinen sowie veränderte Membraneigenschaften sind in der Folge beschrieben worden. Die vor kurzem gemachte Beobachtung, daß verschiedene Glykoproteine der Blutplättchen nicht, wie ursprünglich angenommen, spezifisch für diese Zellen sind, sondern auch in anderen Zellen des strömenden Blutes und der Gefäßwand, wenn auch in etwas veränderter Form, vorkommen können, hat die Frage aufgeworfen, ob als Ursache für die beobachteten Blutungsneigungen nicht auch andere Zellen als ausschließlich die Blutplättchen in Betracht gezogen werden müssen.
As long ago as 1882, Bizzozero recognised the important role that platelets play in thrombosis and haemostasis. Due to their complexity and reactivity, they are difficult cells to work with, and it is only recently that we have begun to understand their physiology and to realise that they are involved in a variety of cellular interactions and pathological states. In their normal non-activated state, platelets are small, anu-cleate discoid cells of 9.5 fl diameter; they are derived from megakaryocytes in the bone marrow, and normally circulate for 9–10 days at a count of 150–400×109/1 (Burstein and Harker 1983). One third of platelets are normally stored in the spleen as an interchangeable pool with circulating cells, and can be pushed into the general circulation in times of stress. Platelet numbers are also increased in acute phase reactions and certain disease states, e.g. myeloproliferative diseases. They are very reactive cells, and on activation by suitable triggers, such as exposure to subendothelial tissues, they are able to adhere at the site of damage, release their contents, aggregate together and form a haemostatic plug. During these processes, platelets also assist fibrin formation by providing a surface on which many of the reactions of the coagulation “cascade” may occur. In a similar way, they later influence control mechanisms via protein C, antithrombin III and fibrinolysis. Many active substances are released: growth factors which influence smooth muscle cells in the vessel wall or tumour growth; serotonin, which affects vascular integrity; vasoactive materials which modulate local blood flow; and leucocyte chemoattractants. If these various functions go out of control, a pathological bleeding or thrombotic state may develop.
Maintenance of blood fluidity and, conversely, arrest of bleeding at sites of vascular injury, depend on complex interactions between clotting factors, platelets, endothelial cells, the subendothelial matrix and proteins of the fibrinolytic pathway. Products generated by one system, e.g., the coagulation cascade, invariably either enhance or inhibit reactions in other pathways. Cleavage of high molecular weight kininogen, for example, amplifies the intrinsic pathway of coagulation and simultaneously releases bradykinin, a peptide with strong vasoconstrictive properties(1). Endothelial cells secrete prostacyclin (PGI2), a potent inhibitor of platelet activity, and both tissue plasminogen activator (t-PA) and t-PA inhibitor, components that play central roles in fibrinolysis(2). In addition, endothelial cells express thrombomodulin, a protein that plays a role in the thrombin-dependent activation of protein C and which ultimately results in the inactivation of Factors Va and VIIIa and release of plasminogen activators(3). Moreover, thrombin generated through the coagulation cascade converts fibrinogen to fibrin, which serves to stabilize developing hemostatic plugs but which also functions as a platelet activating agent.
Direct interactions between cells are essential to the proper functioning of virtually all multicellular organisms. Study of these interactions extends to most disciplines in biology. In addition to bacterial adherence, the subject of this volume, excellent examples are to be found in embryology (interactions in developing tissues), hematology (hemostasis reactions), immunology (lymphocyte and macrophage interactions) and virology (viral infectivity). Although apparently unrelated, each of these processes depends on direct physical interactions between two or more membrane surfaces, and the ability of cells to discriminate between different membranes.
The study of congenital bleeding syndromes has yielded much information on how normal platelets function. In particular, the characterization of the molecular defects in disorders of platelet adhesion and aggregation has resulted in specific roles being proposed for different membrane glycoproteins and α-granule proteins in these processes. In this chapter, we will highlight three major inherited disorders of platelet function: Bemard-Soulier syndrome (BSS), gray platelet syndrome (GPS), and Glanzmann’s thrombasthenia (GT). Each disorder will be introduced by a brief description of the platelet function abnormalities present. This will be followed by a detailed discussion of the specific molecular defects that distinguish the platelets of each syndrome. Emphasis will be placed on reviewing recent data, although an attempt will be made to interpret previous studies in the context of the new advances. Where possible, the different molecular abnormalities will be assessed in terms of how their presence leads to modifications of platelet adhesion and aggregation mechanisms in hemostasis.
The glycoproteins of the platelet plasma membrane, in common with those of the plasma membranes of other cells, have an important role in the interaction of platelets with their environment and in particular in the platelet’s function of hemostasis.
Platelets stop bleeding from damaged blood vessels and initiate repair processes. They contain many important components for these functions, of which surface glycoproteins are critical for two processes, adhesion and aggregation. In platelets, adhesion refers to the attachment of platelets to subendothelium or to other cells, while platelet-platelet “adhesion” is called aggregation to differentiate these processes clearly. Primary adhesion is the binding of resting platelets to subendothelium and secondary adhesion the binding of activated (via partial primary adhesion or temporary association with a thrombus) platelets to subendothelium. Primary adhesion of resting platelets involves several different stages. On initial contact the glycoprotein (GP) Ib-V-IX complex (CD42) binds to von Willebrand factor associated with collagen on the subendothelium surface. The multiple attachments formed stop the platelet, hold it on the surface and start the activation process involving other receptors. The collagen receptor, GPIa-IIa, is an important secondary receptor for platelet adhesion and activation and is critical for inducing the spreading process involving GPIIb-IIIa to assure the intimate contact of the spread platelet with the surface. Other secondary receptors also help to strengthen the links to the surface and act as a reserve in pathological situations. These include fibronectin, laminin and vitronectin receptors. GPIIb-IIIa is particularly important in aggregation and in spreading.
The platelet is arguably the most widely studied of all human cells. It has lent itself well to biochemical investigations, because it is so readily available and can be isolated free from other cell types. Biochemical studies of the platelet have taught us a great deal about amine uptake, storage, and release, lysosomal enzymes, contractile proteins, and the biological significance of cyclic nucleotides. Furthermore, study of the factors involved in platelet adhesion promises to teach us a great deal about the events involved in cell-to-cell and cell-to-substrate contact.
This chapter discusses the structure and function of platelet membrane glycoproteins. Surface glycoproteins are critical for two processes—adhesion and aggregation. Integrins are glycoprotein complexes, consisting of an α- and β-subunit that act as receptors for adhesive proteins present in plasma or on extracellular matrix and link these to the cell cytoskeleton. A common property of integrins is that part of the site they recognize in the adhesive protein is an RGD sequence and peptides related to this sequence can be used to inhibit binding. Specificity of binding to individual adhesive proteins is assured by the conformation of the RGD sequence and by flanking sequences. Platelets are known to contain two integrins of the β3 family, αllbβ3 (GPIIb–IIIa) and αvβ3 (vitronectin receptor), and three of the β1 family, α2βl (GPIa–IIa), α5β1 (GPIc–IIa), and α6β1 (GPIc′–IIa). GPIIb–IIIa are major glycoproteins on the platelet surface with about 50,000 copies/platelet, constituting about 1–2 % of the total platelet protein GPIIb–IIIa can act as a receptor for a number of adhesive proteins including fibrinogen, fibronectin, von Willebrand factor, and possibly thrombospondin and vitronectin. GPIIb-IIIa is normally cryptic in resting platelets and becomes activated and able to bind fibrinogen in activated platelets. GPIa-IIa integrin is also present in much smaller amounts than GPIIb/IIIa on the platelet surface, which is probably related to its role in adhesion and activation instead of aggregation.
Washed human platelets pretreated with chymotrypsin to remove membrane proteins retain full ability to convert 14C-arachidonic acid to thromboxanes. However, the platelets no longer aggregate when treated with arachidonic acid or calcium ionophore A23187. While thromboxane A2 may function as a calcium ionophore to mobilize intraplatelet Ca2+ and to activate platelets for aggregation, the present studies indicate that thromboxane A2 induced platelet aggregation is a process mediated by membrane proteins.
The surface exposed proteins in the range of 43 to 10 kDa were studied with a combination of radiolabelling and two-dimensional electrophoresis. Nineteen proteins were found after reduction, nine of which were linked to other polypeptides in the non-reduced state.
Platelets have been labeled with a lipid soluble complex of the radionuclide indium-111. The complex is formed with 8-hydroxyquinoline and extracted in chloroform which is then evaporated to dryness. The residue is dissolved in 50 μl of ethanol and diluted to 200 μl with normal saline. Platelets are separated by differential centrifugation, washed and suspended either in Tyrodes-albumin solution or in normal saline. The solution of 111In-complex is added to separated platelets and more than 95% of the radioactivity is incorporated with the platelets. The function of the labeled platelets has been studied by their aggregability using adenosine diphosphate and collagen as the stimulating agents. No adverse effects have been observed. The survival of the indium-labeled platelets was similar to that observed with chromium-51 labeled platelets. Labeled canine platelets have been administered to dogs in whom venous thrombi had been induced by alteration of the intima by an electric current. The thrombi were detected by imaging 50 minutes after administration of the labeled platelets. Twenty-four hours later the thrombi were removed and had 20–50 times the radioactivity of an equal weight of blood. Accumulation of large amounts of radioactivity in surgical wounds has also been observed. Damage of the intima of carotid arteries in animals by a balloon catheter demonstrated 8 to 20 times more activity in the damaged artery than in the normal artery. Results indicate that In-111 labeled platelets have potential both for platelet survival studies and for evaluation of vascular damage.
The present status of knowledge about the platelet membrane components suggests that glycoproteins (GP) are involved in the intercellular platelet specific reactions, such as adhesion and aggregation. Normal human platelets and platelet membranes solubilized under dissociating conditions produce characteristic polypeptide and GP electrophoretic patterns (PAGE). Platelets and isolated platelet membranes from patients with chronic myeloid leukemia (CML) presented dissimilarities of their glycoconjugates as compared to the normal platelets. The modification of the electrophoretic banding visualized by the periodic acid-Schiff's reagent (PAS) consisted in the decrease of the 155,000 GP I, the presence of two PAS positive bands in the area of the 135,000 normal PAS positive GP II and a variable decrease of the 100,000 GP III (Apparent mol. wt. are indicated). In addition, preliminary data showed an increased catalytic transfer of the labelled galactosyl and N acetygalactosaminyl residues from exogenous nucleotide 14C-sugar precursors onto the CML-platelet endogenous sugar acceptors. These firstly reported data on molecular abnormalities of the platelet membrane GP in CML, suggested their possible relationship with the impaired platelet adhesion and aggregation occurring in this disease or the ability of leukemic platelets to express modified surface membrane components.
The binding sites for cationized ferritin or ferritin-conjugated wheat germ agglutinin on the cell surface were studied on platelets before or after fixation in glutaraldehyde. The effects of neuraminidase on the binding sites were also demonstrated after fixation of the platelets. Changes in the binding sites and distribution pattern due to exposure to these ligands were further investigated in the unfixed platelets under a variety of conditions such as incubation time and medium. The fixed platelets incubated with either ligand showed an even and continuous distribution of particles on the cell surface. In the unfixed platelets, the ligands were rapidly moved and aggregated probably by lateral migration after binding to the cell surface. The ligands were also bound to the membrane surface and simultaneously appeared in the interior of the open canalicular system. As the binding sites were moved on the cell surface as well as into the open canalicular system, morphological changes suggestive of platelet secretion occurred. The binding sites of either ligand were redistributed on the platelet cell surface. Glycoprotein lb, thought to be the receptor site for wheat germ agglutinin on the cell surface, contains sialic acids that contribute to the negative charge of platelets. Therefore, glycoprotein lb may play an important role as the initial reactive site for thrombotic stimuli.
Whole human platelets and platelet membranes have been solubilized in 1% Triton X-100, and the solubilized proteins examined by crossed immunoelectrophoresis using rabbit antibodies raised against either whole platelets or isolated membranes. 90% of the platelet proteins were solubilized by this extraction. About twenty immunoprecipitates were observed using the extracts obtained from whole platelets, whereas normally eight immunoprecipitates were seen with extracts from isolated membranes. Albumin, factor VIII and fibrinogen were identified with monospecific antibodies.
Correlation of the patterns obtained for platelets or membranes was obtained by addition experiments, by crossed-line immunoelectrophoresis and by crossed immunoelectrophoresis of a mixture of extracts from unlabeled whole platelets and membranes isolated from platelets labeled by lactoperoxidase-catalyzed 125I iodination.
Four sialoglycoproteins were identified by their reduced electrophoretic migration after neuraminidase treatment, and six proteins interacted with various lectins, indicating them to be glycosylated.
Seven amphiphilic proteins were identified by charge-shift crossed immunoelectrophoresis, and nine by crossed hydrophobic interaction immunoelectrophoresis with phenyl-Sepharose.
The topographical arrangement of the membrane proteins was examined with lactoperoxidase-catalyzed 125I-labeled platelets as antigens, and by antibodies absorbed with a suspension of whole platelets. Four and six radioactively labeled precipitates could be identified using the platelet and membrane extracts, respectively, indicating them to be exposed at the outer platelet surface. This was confirmed by the use of antibodies absorbed with intact platelets.
Introduction Proteomics Technology in Nutrition Sciences The Search for Plasma Biomarkers Biomarker Proteins from Blood Cells Mechanistic Biomarkers from Animal Studies Conclusion Acknowledgments References
The binding of phytohemagglutinins from Phaseolus vulgaris (erythroagglutinating phytohemagglutinin (E-PHA) and leukoagglutinating phytohemagglutinin (L-PHA)) and from Lens culinaris (lentil-PHA) to human platelet cell surfaces has been demonstrated. Each platelet binds on an average 500,000 to 600,000 molecules of E-PHA with an apparent dissociation constant of 0.5 x 10⁻⁷m. These values were 250,000 to 350,000 molecules bound for L-PHA and 300,000 to 400,000 molecules bound for lentil-PHA with dissociation constants of 4 x 10⁻⁷m and 1.4 x 10⁻⁷m, respectively. Both E-PHA and lentil-PHA can be released from the platelet surface using appropriate oligosaccharide haptene inhibitors, indicating that the phytohemagglutinins do not enter the cells. When E-PHA and L-PHA bind to platelets the cells are aggregated, adenylate cyclase is inhibited, and a protein designated as thrombin-sensitive protein is released from the particulate fraction of the cell. Thus, binding of these compounds to the platelet surface mimics thrombin-induced aggregation and the release reaction. Lentil-PHA binds tightly to platelets but the cells do not aggregate and there is no inhibition of adenylate cyclase or release of thrombin-sensitive protein. Thrombin induces platelet aggregation, adenylate cyclase inhibition, and thrombin-sensitive protein release even when platelets are saturated with lentil-PHA. After incubation of platelets with either thrombin or L-PHA, there is an apparent 2-fold increase in the number of receptor sites for lentil-PHA, suggesting that these compounds produce a conformational change in the platelet surface exposing increased numbers of lentil-PHA receptor sites.
Inbar et al.21 reported that tumour cells are not agglutinable with Con A at low temperature (4° C), but regain their usual Con A agglutination properties upon warming to room temperature (24° C) without an increase in Con A sites. Although they interpreted this as evidence of two distinct types of Con A surface receptors ("A sites” or Con A agglutination sites and “B sites” or Con A binding sites21), their data can be interpreted in terms of the suggestion18 that low temperature does not allow significant lateral movement of the Con A sites into clusters necessary to the formation of multiple cross links between agglutinated cells.
The antibiotic ristocetin, in concentrations of 1.0-1.5 mg/ml, aggregated normal platelets in citrated platelet-rich plasma by a mechanism in which the release reaction played only a minor role. Platelet aggregation by ristocetin in a concentration of 1.2 mg/ml was absent or markedly decreased in 10 patients with von Willebrand's disease. Lesser degrees of abnormality were obtained with a concentration of 1.5 mg/ml. The magnitude of the defect in ristocetin-induced platelet aggregation correlated well with the degree of abnormality of the bleeding time and the levels of antihemophilic factor (AHF, VIII(AHF)) procoagulant activity. In all patients, the defect in ristocetin-induced platelet aggregation was corrected in vitro by normal plasma. Correction was also obtained with a fraction of normal cryoprecipitate that eluted in the void volume with VIII(AHF) after chromatography on a gel that excludes molecules larger than 5 x 10(6). A similar fraction, devoid of VIII(AHF) activity, obtained from patients with von Willebrand's disease had no corrective effect, but fractions obtained from patients with hemophilia were just as effective as those obtained from normal subjects. The correction activity of plasma and partially purified factor VIII was inhibited by a rabbit antibody to human factor VIII but not by a human antibody against VIII(AHF) procoagulant activity. The studies provide further evidence that patients with von Willebrand's disease are deficient in a plasma factor that is necessary for normal platelet function. The activity of this factor appears to be associated with factor VIII but is unrelated to VIII(AHF) procoagulant activity.
Isolated human platelet membranes which have been analyzed by sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis have been shown to contain a heterogeneous group of polypeptides of different molecular weight classes ranging from 10,000 to over 250,000. Three major glycoproteins have been identified in the platelet membrane. Some of the architectural relationships of the platelet membrane polypeptides were probed by analysis of the residual protein gel patterns following incubation of the intact cells with proteolytic enzymes. The membrane polypeptides in their native state in intact cells were relatively resistant to low dose Pronase digestion in contrast to the high sensitivity of the isolated membrane fragments to enzyme digestion. The membrane glycoproteins in intact cells were particularly susceptible to trypsin and papain action. Acetylcholinesterase was a readily accessible membrane constituent which was rapidly inactivated by small amounts of Pronase. N-Ethyl[¹⁴C]maleimide and [²⁰³Hg]p-chloromercuribenzene sulfonate labeling of intact whole platelets revealed three major membrane polypeptides with reactive sulfhydryl groups. Isotopic labeling of one of these membrane polypeptides was partially blocked by prior incubation of washed platelets with anti-F(ab′)2 thrombosthenin fragments.
Present methods for assay of platelet aggregating agents use freshly prepared platelets. Much time is spent in daily preparation of platelets and standardization presents problems. The preparation of fixed washed platelets (FWP) and their use in two bioassays are described in this report. Washed human platelets were fixed for 48 hours with 4 per cent paraformaldehyde, washed twice in phosphate buffer, pH 6.4, and stored at 4 degrees C. Aggregation of FWP was studied with a macroscopic test and a light absorbance measurement. FWP did not aggregate with adenosine diphosphate, collagen, adrenalin, and thrombin. FWP aggregated with bovine or porcine plasma, poly-L-lysine, and ristocetin with normal human plasma but not with von Willebrand's disease plasma. These observations confirm the direct aggregating effect of these agents. Macroscopic aggregation times were dependent on the amount of aggregating agent (bovine plasma, normal human plasma). A quantitative assay for bovine platelet aggregating factor (PAF) and von Willebrand factor (vWF) with FWP was developed. The ability of FWP to aggregate remained unchanged after 1 month of storage at 4 degrees C. Ristocetin alone caused a decrease in light transmission of FWP suspensions, depending upon the concentration of ristocetin, but did not cause aggregation. FWP constitute a stable reagent suitable for quantitative measurement of PAF and vWF.
Separation of platelets from plasma is achieved by adding ADP (final concentration 10-5 M) to platelet-rich plasma and allowing aggregates to form. Aggregates are removed quickly by brief, gentle centrifugation, washed two to three times with 0.9% NaCl (saline), and then incubated for 10 minutes in the presence of apyrase, albumin and calcium. Platelet aggregates deaggregate completely during this incubation period. The platelet suspension is then subjected to 1100g for 12 minutes, gently resuspended in a small volume of saline, and finally diluted with an appropriate medium to the desired concentration. The entire separation procedure requires approximately 30 minutes. Platelets obtained by this procedure are a) comparable in aggregability to the platelet preparations obtained by gel filtration, b) have normal intracellular amounts of ATP and ADP, and c) except for slight dilatation of the surface-connected canalicular system, have normal ultrastructural appearance. When suspended in an appropriate medium, these separated platelets take up serotonin 14-C and subsequently release it in nearly normal quantities when exposed to thrombin, collagen or ADP.
THE haemostatic role of the blood platelet is largely dependent on its
ability to adhere to exposed subendothelial components in the event of
vessel damage1 and respond to specific aggregation-inducing
stimuli, such as adenosine diphosphate (ADP), which seem to act at
defined receptor sites on the platelet membrane2. The early
stages of platelet adhesion and aggregation are surface-mediated
phenomena and much interest has centred on determining which of the
surface groupings are involved in these mechanisms. Iodination
techniques3,4 have revealed that a limited number of proteins
are exposed on the surface of the platelet and that among these are the
major membrane glycoproteins. A possible involvement of carbohydrate
groupings in the mechanisms of aggregation as induced by ADP and
5-hydroxytryptamine was described by Mester et al.5 who
showed that the velocity of aggregation was altered by changes in the
sialic acid content of the platelet.
THE platelet plasma membrane contains several proteins of different molecular weights. Three of these proteins stain for carbohydrate with periodic acid-Schiff reagent and have been termed glycoproteins I, II and III (molecular weights 150,000, 124,000 and 106,000, respectively)1,2. It has been suggested that the bleeding tendency observed in Glanz-mann's thrombasthenia results from abnormalities of the platelet membrane3-6. Nurden and Caen7 showed that glycoprotein II was absent in a crude membrane fraction isolated from thrombasthenic platelets. We have now investigated the surface composition of thrombasthenic membranes by the lactoperoxidase iodination technique2 and found a low concentration of glycoprotein II, and other molecular differences.
The exposed proteins of the plasma membrane of normal human lymphocytes and platelets were labelled by using the lactoperoxidase macromolecular probe system. The labelled components were separated into molecular-weight classes by sodium dodecyl sulphate--polyacrylamide-gel electrophoresis. In contrast with the report by Tanner et al. (1974), a comparison of the two cell types showed that the major labelled components in both cell types were glycoproteins and were not identical. It is concluded that the exposed proteins are probably the most distinguishing characteristic of the plasma membrane of differentiated cell types.
The platelets from two related patients with the hereditary giant platelet syndrome were examined. They were larger than normal but otherwise ultrastructurally normal; they contained increased storage pools of adenine nucleotides and heparin-neutralizing activity and took up serotonin at an increased rate. They aggregated normally with ADP and collagen but failed to aggregate with bovine factor VIII and Ristocetin. Some change in shape occurred with ADP, and the reduction in adenylate energy change after addition of ADP to platelet-rich plasma was smaller than normal. Platelet coagulant activities including contact product forming activity, intrinsic factor-Xa forming activity and platelet factor 3 activity were normal or increased, but collagen-induced coagulant activity was absent whether tested in washed platelet suspensions or platelet-rich plasma. Platelet washing experiments showed decreased binding of factors V and VIII to hereditary giant platelets and no detectable factor XI in washed platelet suspensions. It is concluded that (1) the hereditary giant platelets studied lacked a binding mechanism for factors, V, VIII and XI; (2) the normal development of collagen-induced coagulant activity apparently depends upon the binding of factor XI to the platelet membrane; and (3) the defective prothrombin consumption observed in these patients may have resulted from the failure of their platelets to bind factor XI.
Ristocetin induces aggregation of normal platelet rich plasma over a wide range of concentrations. Low doses induce a biphasic response of which the first wave is not mediated by ADP and proceeds without the initial platelet shape change. The absence of aggregation in von Willebrand's disease results from the lack of a component, Willebrand factor, which is eluted together with platelets, factor VII activity and Willebrand (or factor VIII related) antigen in the void volume fraction when platelet rich plasma is gel filtered (Sepharose 2B). Twice washed normal platelet aggregate to ristocetin whereas 4 times washed platelets only aggregate when low volumes of normal haemophilia A plasma of serum are added. The interaction of ristocetin with platelets and plasmatic components has been investigated. A mechanism is proposed for ristocetin induced aggregation.
Intact or isolated plasma membranes of human platelets were treated with thrombin (EC 3.4.4.13), and the membrane proteins hydrolyzed by this enzyme were identified by acrylamide gel electrophoresis and lactoperoxidase-catalyzed iodination. In isolated membranes, a high-molecular-weight polypeptide (220 000) and three glycoproteins with molecular weights of 150 000, 118 000 and 93 000 were reduced in concentration and/or molecular weight after thrombin treatment. In intact membranes, however, only the 118 000-mol. wt glycoprotein was reduced in concentration. Although two other thrombin substrates (the 150 000- and 93 000-mol. wt glycoproteins) were exposed on the surface of intact membranes, they were not hydrolyzed. Thus, the plasma membrane of platelets appears to contain at least two thrombin substrates which because of their orientation in the membrane, are resistant to hydrolysis on intact platelets. The 118 000-mol. wt glycoprotein, the only protein hydrolyzed by thrombin on intact platelets, may be the proteolytic site of thrombin action on the membrane surface.
The topographical distributions of concanavalin A-binding sites on the surfaces of 3T3, proteasetreated 3T3, and simian virus 40-transformed 3T3 cultured mouse fibroblasts appear to be different, as shown by a shadow-cast replica technique using concanavalin A and a hemocyanin marker, or as shown previously on isolated membranes with concanavalin A coupled to ferritin. However, chemical fixation of cells before labeling with concanavalin A and hemocyanin, or labeling exclusively at 4 degrees , allows one to distinguish between inherent concanavalin A-binding-site topography and potential rearrangement of sites induced by the action of the multivalent concanavalin A molecule itself. The inherent distribution of binding sites on 3T3, protease-treated 3T3, and transformed cells is actually the same on all cells, i.e., dispersed and random. Treatment of unfixed transformed or protease-treated 3T3 cells, but not normal 3T3 cells, with concanavalin A and hemocyanin at 37 degrees (or at 4 degrees with subsequent warming to 37 degrees ), however, results in clustering of binding sites, presumably due to crosslinking of neighboring lectin-binding sites by the quadrivalent concanavalin A. Thus, the underlying difference between concanavalin A-binding sites on normal as compared with transformed or protease-treated normal cells lies not in the inherent topography of binding sites, but rather in the susceptibility of the sites to aggregation by concanavalin A. The latter may reflect an increased mobility of lectin-binding sites on transformed or protease-treated cells.
The arrangement of proteins in the outer membrane of rat liver mitochondria was explored. Exterior membrane proteins of the mitochondrion were labeled with 125I using lactoperoxidase-catalyzed iodination. The iodine label thus served as a marker for outer membrane proteins. The specific activity of the outer membrane was 12-fold greater than that of the mitochondria from which it was isolated by digitonin treatment. Adenylate kinase, a soluble enzyme located between the outer and inner membranes, was assayed at various stages in the experiments to determine whether or not the mitochondria were intact. After iodination of the mitochondria the polypeptides of the organelles were separated into molecular weight classes by electrophoresis in a sodium dodecyl sulfate-acrylamide gel system. When intact mitochondria were iodinated, the label was distributed among polypeptides in 12 molecular weight classes. In microsomes the label was found in polypeptides of nine molecular weight classes. Although the iodinated polypeptides of both mitochondria and microsomes ranged from about 10,000 to 100,000 daltons, the radioactive gel patterns of the two organelles were quite different. About 70% of the label in the outer membrane isolated from the iodinated mitochondria was in polypeptides of the 14,000 molecular weight class. The distribution of the labeled polypeptides in the various molecular weight classes was different for proteins in isolated outer membrane fragments as compared to proteins remaining with the inner membrane matrix particles after the digitonin fractionation. This selective fractionation of outer membrane proteins suggested that the proteins were heterogeneously distributed in the plane of the membrane.
1.1. The following lectins have been studied with regard to their effects on platelet functions: wheat germ agglutinin (), Phaseolus coccineus (determinant unknown), Ricinus communis and Agaricus bisporus (galactose determinant), concanavalin A, Lathyrus sativus, Lens culinaris and Pisum sativum (glucose, mannose determinants), and soybean lectin ().2.2. Soybean lectin ) was virtually without effect on platelet aggregation or release.3.3. Lectins with glucose and mannose specificities had litle effect on platelets but, at high concentrations, did induce some release of serotonin but not of ADP, suggesting two different mechanisms for the release of these two constituents.4.4. Lectins with specificities towards galactose (especially R. communis), (wheat germ agglutinin) and the lectin of P. coccineus (determinant unknown) were strong inducers of platelet aggregation and the release of both ADP and serotonin; the aggregation induced by R. communis was inhibited by the destruction of ADP while that induced by wheat germ agglutinin and P. coccineus was not greatly inhibited, suggesting that these may act as true agglutinins. At subthreshold concentrations, these lectins also inhibited aggregation induced by ADP, and serotonin release induced by collagen. These lectins also induced the agglutination of platelets and platelet membranes in a microtiter assay system.5.5. Concanavalin A (glucose, mannose determinant) was unusual in that it caused agglutination only of membranes and not of intact platelets but did cause release of ADP and serotonin. At subthreshold concentrations, it also inhibited the release of serotonin by collagen.6.6. The agglutination of platelet membranes by wheat germ agglutinin and P. coccineus was specifically inhibited by the macroglycopeptide (Glycopeptide I) of human platelets and by platelet Glycopeptide II, respectively. Platelet Glycopeptide III was without effect on any of the lectins studied.
The ionophores A 23187 and X-537 A induce an uptake of 45Ca by human blood platelets. They induce the release of adenine nucleotides and of serotonin. A 23187 also induces platelet aggregation and the retraction of a clot formed in platelet-rich plasma by reptilase. These results suggest that an increase of the concentration of Ca2+ in the cytoplasm plays a role in the activation of blood platelets.
A glycoprotein abnormality in membrane fractions isolated from the platelets of patients with Glanzmann's thrombasthenia is described. Glycoproteins solubilized from thrombasthenic and control human platelets have been compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Whereas in a control human platelet membrane fraction three major glycoproteins of molecular weight 155 000, 135 000 and 103 000 were observed, in a similar fraction from thrombasthenic platelets a glycoprotein of 152 000 molecular weight predominated. No differences were observed in the nature of a high molecular weight acidic glycopeptide released from washed thrombasthenic and control platelet suspensions by trypsin, and the sialie acid content of thrombasthenic platelets was shown to be within the normal range.
It is suggested that the glycoprotein abnormality may be due to an altered organization within the thrombasthenic platelet membrane and that further studies are necesasry to investigate the role of membrane glycoproteins in platelet function.
The platelets of two patients with the Bernard-Souller (giant platelet) syndrome were not aggregated by bovine factor VIII. Platelet aggregation by rlstocetin was also absent and, in contrast to the findings in von Willebrand's disease, this defect was not corrected by human factor VIII. The platelets of patients with the Bernard-Soulier syndrome may lack a receptor for the von Willebrand factor activity of factor VIII (VIIIVWF), whereas the abnormal platelet function in von Willebrand's disease is due to a decreased level of VIIIVWF in their plasma. As in the latter disorder, the adhesion of platelets to subendothellum was impalred in the two patients with the Bernard-Souller syndrome that we studied. These findings provide further evidence that VIIIVWF, through its effect on platelets, plays an important role during the primary arrest of bleeding.
The addition of 5% E isomer to a sample of synthetic pheromone [(Z) 8 dodecen 1 ol acetate containing 1.7% E isomer] plus synergist, increased activity 25 times. Tests were conducted in a commercial peach orchard from August 10 to 28, 1972, with the Zoecon Pherotrap 1C (bottom and top sections), using as bait a 43x5.3x1 mm section of rubber band containing a mixture of 2 μl of dodecyl alcohol and 0.2 μl of 8 dodecen 1 ol acetate with one of the following isomeric compositions: 98.3% Z+1.7% E, 93.3% Z+6.7% E, and 83.3% Z+16.7% E. (Isomer composition was determined by capillary gas chromatography with a SCOT column, 0.5 mm inner diameter, containing silicone polymer EGSS X at 135° C and a helium flow rate of 4 ml min-1.) Captures of male moths during the 19 d test period, totalled 20, 505, and 1, respectively, in the four replicates of each treatment. Repetition of the experiment (albeit late in the season when the moth population was low) to define more precisely the optimum amount of E isomer, resulted in the following moth captures with the indicated percentages of E isomer in the pheromone: 5 with 1.7%, 23 with 4.2%, 26 with 6.7%, 34 with 9.2%, 28 with 11.7%. The optimum amount of E isomer seems to be about 8%. (In a concurrent cooperative study, a similar enhancement effect was observed with the synthetic pheromone of the European corn borer and the redbanded leafroller). It is interesting that the E isomer is reported to inhibit attraction of the oriental fruit moth.
The platelets of three patients with the hereditary giant platelet syndrome of Bernard and Soulier failed to aggregate in response to either ristocetin or bovine fibrinogen. The results of aggregation experiments using mixtures of platelets and plasma suggest that a reaction between a plasma factor deficient in von Willebrand's disease and a platelet component lacking in our patients, and leading to platelet aggregation independently of adenosine diphosphate (ADP), is essential for normal haemostasis.
It is known that Ristocetin acts as an aggregating agent of normal platelets, but in the presence of Von Willebrand plasma, platelets do not aggregate under the influence of Ristocetin. In addition, anti human Factor VIII antibody blocks Ristocetin effect in normal plasma. The authors demonstrate that Bernard Soulier platelets do not aggregate on exposure to Ristocetin in any plasma, whether normal, hemophiliac, or Von Willebrand. They postulate that a Ristocetin Von Willebrand receptor is lacking in Bernard Soulier platelets. They further suggest that a vascular wall component other than collagen (possibly microfibrils or basement membrane) might function in the same manner as Ristocetin in primary hemostasis, resulting in Factor VIII activation and platelet aggregation via Von Willebrand factor interaction. (Van Slyck - Detroit, Mich.)
IT has been reported1 that Ristocetin induces aggregation of
platelets in normal platelet-rich plasma (PRP) but not of platelets from
patients with von Willebrand's disease. Results presented here
demonstrate that the aggregation of platelets induced by Ristocetin
involves a factor present in normal and haemophilia A plasma and absent
in Willebrand plasma. Lack of such a factor would then explain the
inability of platelets to aggregate in response to Ristocetin in von
Wille-brand's disease. In eight patients fulfilling the criteria of von
Willebrand's disease (Table 1), platelet aggregation induced by
Ristocetin was markedly deficient when compared to healthy normal
plasmas or did not occur at all. The abnormal responses of platelets
from von Willebrand's patients could be corrected by the addition of
small volumes of normal platelet-poor plasma (PPP) (Fig. 1); similar
correction was achieved by Haemophilia A PPP but not by PPP from
patients with von Willebrand's disease.
The adhesion of platelets in flowing blood to the subendothelial surface of rabbit aorta was studied by a quantitative, in vitro method. Platelet adhension was significantly decreased in 5 patients with von Willebrand's disease. Impaired adhesion of platelets to some component of the blood vessel wall may account for the prolonged bleeding time in these patients.
. Two methods of assay of Willebrand factor activity have been used in the study of 20 patients with congenital and acquired von Willebrand's disease. The relationship between the three entities of factor-VIII complex, Willebrand factor activity, Willebrand antigen and factor-VIII activity is discussed. Data from thermo-stability and dissociation studies favour the hypothesis that factor-VIII complex consists of two closely related but separate proteins.
The polypeptides and glycoproteins in the human platelet plasma membrane were analyzed by polyacrylamide disc gel electrophoresis containing sodium dodecyl sulfate. Protein stains of these gels showed that this membrane is composed of polypeptides of molecular weights varying from more than 200,000 to 13,000. It was also shown that this membrane contains three major glycoproteins. The molecular weights of these glycoproteins were determined to be 150,000, 118,000, and 92,000, respectively. The membrane proteins that have exposure on the membrane surface were determined by the lactoperoxidase iodination technique. Use of this technique will iodinate only those membrane proteins which exist on the exposed surface of the plasma membrane. It was shown that at least seven different membrane polypeptides were labeled and are therefore exposed to the outside of the platelet. These labeled polypeptides correspond to the three major glycoproteins in the membrane in addition to four polypeptides with lower molecular weights (68,000, 34,000, 18,000, and 13,000, respectively). Alterations induced by trypsin on the plasma membrane surface were also in-vestigated. Of the iodinated surface components, only the molecular weights of the three glycoproteins were decreased by this proteolytic enzyme, suggesting that the primary target for proteolytic enzymes on the membrane is glycoprotein. The glycopeptide hydrolytic products that remain with the membrane all contain iodine indicating that the iodinatable tyrosine residue on all three glycoproteins is on the membrane side of the trypsin-sensitive bond. Iodination of trypsin-treated platelets showed that the glycopeptide hydrolytic products are no longer in an iodinatable position in the membrane, demonstrating that hydrolysis of exposed glycoprotein will induce a conformational change in the plasma membrane. Trypsin hydrolysis was also shown to decrease the concentration of a polypeptide with a molecular weight of 150,000. However, this polypeptide does not appear to be a plasma membrane component since it is not isolated with the membrane fraction nor iodinated by lactoperoxidase. It therefore appears that it is released from within the platelet as a result of glycoprotein hydrolysis on the membrane surface.
:Three patients with congenital macrothrombocytic thrombocytopenia are described. The platelets had a reduced electrophoretic mobility, and the content of sialic acid was also abnormally low, suggesting a defect in the platelet membrane. Platelets were rapidly removed from the circulation, possibly because the platelet membrane defect was associated with reduced electrostatic repulsive forces.
1. The antibotic, Ristocetin, causes precipitation of fibrinogen from platelet poor plasma, as well as platelet aggregation.
2. The precise mechanism of Ristocetin’s effect on platelets has not been elucidated, but it has been shown to initiate ADP release, which may contribute in part to its aggregating ability.
3. Ristocetin has been shown to produce aggregation in platelet rich plasma from thrombasthenic patients.
4. Three patients with Von Willebrand’s disease were examined. In 2, Ristocetin caused no platelet aggregation whatsoever, whilst the third aggregated normally. It is suggested, on this basis, that Von Willebrand’s disease may be subdivided into two types and that Ristocetin could prove to be a valuable technique for further study of this group of disorders.
The action of thrombin on fibrinogen is not responsible for its effect on platelets. Thrombin does, however, have an enzyme effect on a platelet constituent: this requires an unsubstituted serine hydroxyl group and a group susceptible to acetylation.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.