Sriram Krishnaswamy

Sriram Krishnaswamy
University of Pennsylvania | UP · Department of Pediatrics

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102
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Publications

Publications (102)
Article
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The proteolytic conversion of factor V to factor Va is central for amplified flux through the blood coagulation cascade. Heterodimeric factor Va is produced by cleavage at three sites in the middle of factor V by thrombin yielding an N-terminus-derived heavy chain and a C-terminus-derived light chain. Here, we show that light chain formation result...
Article
Factor VIII (FVIII) has a multi-domain structure (A1-a1-A2-a2-B-a3-A3-C1-C2), and intracellular processing within the B domain results in its secretion as a heterodimeric procofactor consisting of a variably sized heavy chain (A1-a1-A2-a2-B) and a light chain (a3-A3-C1-C2). Proteolytic cleavage by thrombin at R372, R740, and R1689 removes the B-dom...
Article
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Unfractionated heparin (UFH), the standard anticoagulant for cardiopulmonary bypass (CPB) surgery, carries a risk of post-operative bleeding and is potentially harmful in patients with heparin-induced thrombocytopenia–associated antibodies. To improve the activity of an alternative anticoagulant, the RNA aptamer 11F7t, we solved X-ray crystal struc...
Article
Full-text available
This 9th Symposium on Hemostasis is an international scientific meeting held biannually in Chapel Hill, North Carolina. The meeting is in large measure the result of the close friendship between the late Dr. Harold R. Roberts of UNC Chapel Hill and Dr. Ulla Hedner of Novo Nordisk. When Novo Nordisk was developing the hemophilia therapy that would b...
Article
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Platelets are increasingly recognized for their contributions to tumor metastasis. Here, we show that the phosphoinositide signaling modulated by phosphatidylinositol transfer protein type α (PITPα), a protein which shuttles phosphatidylinositol between organelles, is essential for platelet-mediated tumor metastasis. PITPα-deficient platelets have...
Article
Safe and effective antithrombotic therapy requires understanding of mechanisms that contribute to pathological thrombosis but have lesser impact on hemostasis. We found that the extrinsic tissue factor (TF) coagulation initiation complex can selectively activate the anti-hemophilic cofactor, FVIII, triggering the hemostatic intrinsic coagulation pa...
Article
Direct inhibitors of coagulation factor Xa (FXa) or thrombin are promising oral anticoagulants that are becoming widely adopted. The ability to reverse their anticoagulant effects is important when serious bleeding occurs or urgent medical procedures are needed. Here, using experimental mouse models of hemostasis, we show that a variant coagulation...
Chapter
Thrombin formation is essential for clot formation and regulated hemostatic response. In this chapter, we outline mechanistic features of the reactions at play in the activation of its zymogen precursor and the resultant functions of thrombin. These features represent an exemplar for the mechanisms underlying the other membrane-dependent reactions...
Article
Full-text available
Thrombin is produced from the C-terminal half of prothrombin following its proteolytic activation. The N-terminal half, released as the propiece Fragment 12 (F12), is composed of an N-terminal γ-carboxyglutamate domain (Gla) followed by two kringles (K1 and K2). The propiece plays essential roles in regulating prothrombin activation and proteinase...
Conference Paper
Distinctive protein substrate specificities are achieved by the membrane-bound enzyme complexes of coagulation, despite the comparable active site geometries of the structurally homologous, trypsin-like coagulation proteinases. In contrast to long-standing ideas in the field, there is increasing evidence for the prevalence of a binding strategy in...
Article
Distinctive protein substrate specificities are achieved by the membrane-bound enzyme complexes of coagulation, despite the comparable active site geometries of the structurally homologous, trypsin-like coagulation proteinases. In contrast to long-standing ideas in the field, there is increasing evidence for the prevalence of a binding strategy in...
Article
Phosphatidylinositol (PtdIns) is a relatively rare phospholipid in cell membranes. However, because of its unique ability to be transiently phosphorylated, it is critical for platelet signaling and vascular plug formation. Phosphatidylinositol transfer proteins (PITPs) facilitate the transfer of aqueous insoluble phosphatidylinositol in vitro from...
Article
In this issue of Blood , back-to-back (dos-a-dos) papers by [Chiu et al][1][1][2] and [Yee et al][3][2][4] present complementary findings of structural investigations into the interaction between factor VIII (FVIII) and von Willebrand factor (VWF). The binding of FVIII to VWF contributes in a major
Article
Hemorrhagic complications may occur during anticoagulant therapy, but clinical studies indicate that FXa-directed anticoagulants are not inferior to vitamin K antagonists in preventing thrombosis while decreasing the incidence of intracranial bleeding. Anti-hemophilic FVIII has a central role in hemostasis, thus we hypothesized that elucidating eff...
Article
The membrane-dependent interaction of factor Xa (FXa) with factor Va (FVa) forms prothrombinase and drives thrombin formation essential for hemostasis. Activated platelets are considered to provide the primary biological surface to support prothrombinase function. However, the question of how other cell types may cooperate within the biological mil...
Article
Histones are detrimental in late sepsis. Both activated protein C (aPC) and heparin can reverse their effect. Here, we investigated whether histones can modulate aPC generation in a manner similar to another positively charged molecule, platelet factor 4, and how heparinoids (unfractionated heparin or oxygen-desulfated unfractionated heparin with m...
Article
Full-text available
Coagulation factor V (FV) circulates as an inactive procofactor and is activated to FVa by proteolytic removal of a large inhibitory B-domain. Conserved basic and acidic sequences within the B-domain appear to play an important role in keeping FV as an inactive procofactor. Here, we utilized recombinant B-domain fragments to elucidate the mechanism...
Article
Full-text available
Long-standing dogma proposes a profound contribution of membrane binding by prothrombin in determining the rate at which it is converted to thrombin by prothrombinase. We have examined the action of prothrombinase on full-length prothrombin variants lacking γ-carboxyglutamate modifications (desGla) with impaired membrane binding. We show an unexpec...
Article
The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root...
Article
The proteolytic conversion of prothrombin to thrombin catalyzed by prothrombinase is one of the more extensively studied reactions of blood coagulation. Sophisticated biophysical and biochemical insights into the players of this reaction were developed in the early days of the field. Yet, many basic enzymological questions remained unanswered. I su...
Article
Crystal structures of factor (F) VIIa/soluble (s) tissue factor (TF), obtained under high Mg2+ (50mM Mg2+/5mM Ca2+), have three of seven Ca2+ sites in the γ-carboxyglutamic acid (Gla) domain replaced by Mg2+ at positions 1, 4, and 7. We now report structures under low Mg2+ (2.5mM Mg2+/5mM Ca2+) as well as under high Ca2+ (5mM Mg2+/45mM Ca2+). Under...
Article
Full-text available
Thrombin is produced by the ordered action of prothrombinase on two cleavage sites in prothrombin. Meizothrombin, a proteinase precursor of thrombin, is a singly cleaved species that accumulates abundantly as an intermediate. We now show that covalent linkage of the N-terminal propiece with the proteinase domain in meizothrombin imbues it with exce...
Article
In this issue of Blood,Olson et al establish the biochemical basis for the specificity and selectivity of a dual-action anticoagulant with the designed feature of rapid neutralization, making it extremely attractive for use in anticoagulated patients if surgery is warranted.
Article
Significant gaps remain in the understanding of how blood cells and the vasculature differentially support coagulation enzyme complex function leading to regulated thrombus formation in vivo. While studies employing knock-out or transgenic mice have proved useful many of these scientific gaps partly result from the lack of molecular approaches and...
Article
Full-text available
Heparin-induced thrombocytopenia (HIT) is caused by antibodies that recognize complexes between platelet factor 4 (PF4) and heparin or glycosaminoglycan side chains. These antibodies can lead to a limb- and life-threatening prothrombotic state. We now show that HIT antibodies are able to inhibit generation of activated protein C (aPC) by thrombin/t...
Article
Mouse and human prothrombin (ProT) active site specifically labeled with d-Phe-Pro-Arg-CH2Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating...
Article
Full-text available
Mouse and human prothrombin (ProT) active site specifically labeled with d-Phe-Pro-Arg-CH2Cl (FPR-ProT) inhibited tissue factor-initiated thrombin generation in platelet-rich and platelet-poor mouse and human plasmas. FPR-prethrombin 1 (Pre 1), fragment 1 (F1), fragment 1.2 (F1.2), and FPR-thrombin produced no significant inhibition, demonstrating...
Article
Full-text available
The critical and multiple roles of thrombin in blood coagulation are regulated by ligands and cofactors. Zymogen activation imparts proteolytic activity to thrombin and also affects the binding of ligands to its two principal exosites. We have used the activation peptide fragment 1.2 (F12), a ligand for anion-binding exosite 2, to probe the zymogen...
Article
Full-text available
The interaction of factor Xa with factor Va on membranes to form prothrombinase profoundly increases the rate of the proteolytic conversion of prothrombin to thrombin. We present the characterization of an RNA aptamer (RNA11F7t) selected from a combinatorial library based on its ability to bind factor Xa. We show that RNA11F7t inhibits thrombin for...
Article
Full-text available
Prothrombinase converts prothrombin to thrombin via cleavage at Arg320 followed by cleavage at Arg271. Exosite-dependent binding of prothrombin to prothrombinase facilitates active site docking by Arg320 and initial cleavage at this site. Precise positioning of the Arg320 site for cleavage is implied by essentially normal cleavage at Arg320 in reco...
Article
Full-text available
In this issue of Blood, Undas and colleagues show that patients with AMI are able to mount an enhanced blood clotting response in the peripheral microvasculature.
Article
Full-text available
Membrane binding by prothrombin, mediated by its N-terminal fragment 1 (F1) domain, plays an essential role in its proteolytic activation by prothrombinase. Thrombin is produced in two cleavage reactions. One at Arg320 yields the proteinase meizothrombin that retains membrane binding properties. The second, at Arg271, yields thrombin and severs cov...
Article
Full-text available
Proteolytic processing of von Willebrand factor (VWF) by ADAMTS13 metalloproteinase is crucial for normal hemostasis. In vitro, cleavage of VWF by ADAMTS13 is slow even at high shear stress and is typically studied in the presence of denaturants. We now show that, under shear stress and at physiological pH and ionic strength, coagulation factor VII...
Article
Full-text available
The preferred pathway for prothrombin activation by prothrombinase involves initial cleavage at Arg(320) to produce meizothrombin, which is then cleaved at Arg(271) to liberate thrombin. Exosite binding drives substrate affinity and is independent of the bond being cleaved. The pathway for cleavage is determined by large differences in V(max) for c...
Article
Full-text available
Prothrombinase catalyzes thrombin formation by the ordered cleavage of two peptide bonds in prothrombin. Although these bonds are likely ≈36 Å apart, sequential cleavage of prothrombin at Arg-320 to produce meizothrombin, followed by its cleavage at Arg-271, are both accomplished by equivalent exosite interactions that tether each substrate to the...
Article
During blood coagulation, factor IXa (FIXa) activates factor X (FX) requiring Ca2+, phospholipid, and factor VIIIa (FVIIIa). The serine protease domain of FIXa contains a Ca2+ site and is predicted to contain a Na+ site. Comparative homology analysis revealed that Na+ in FIXa coordinates to the carbonyl groups of residues 184A, 185, 221A, and 224 (...
Article
Full-text available
The interaction of thrombin (IIa) with thrombomodulin (TM) is essential for the efficient activation of protein C (PC). Interactions between PC and extended surfaces, likely contributed by TM within the IIa.TM complex, have been proposed to play a key role in PC activation. Initial velocities of PC activation at different concentrations of PC and T...
Article
Macromolecular substrate recognition and serine proteinase specificity lie at the heart of the tightly regulated hemostatic response. Mechanisms established for the less specific serine proteinases of digestion have played a dominant role in guiding investigations of the basis for the narrow specificities exhibited by the coagulation enzymes. These...
Article
Full-text available
Thrombin formation results from cleavage of prothrombin following Arg(271) and Arg(320). Both bonds are accessible for cleavage, yet the sequential action of prothrombinase on Arg(320) followed by Arg(271) is implied by the intermediate observed during prothrombin activation. We have studied the individual cleavage reactions catalyzed by prothrombi...
Article
A prothrombin mutant that is activated to yield stable meizothrombin produces dominant inhibition of clot formation in a mouse model of carotid artery injury. Studies of clot formation in vivo that draw from established details of coagulation biochemistry have the potential for yielding unexpected
Article
Full-text available
The conversion of prothrombin to thrombin is catalyzed by prothrombinase, an enzyme complex composed of the serine proteinase factor Xa and a cofactor protein, factor Va, assembled on membranes. Kinetic studies indicate that interactions with extended macromolecular recognition sites (exosites) rather than the active site of prothrombinase are the...
Article
Full-text available
The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigate...
Article
The activation of factor X by VIIa/TF and the Xa-dependent inhibition of the enzyme complex by tissue factor pathway inhibitor (TFPI) are considered primary steps in the initiation of coagulation. IX activation by VIIa/TF is considered to contribute catalyst necessary for further Xa production in the ensuing amplification phase. We have investigate...
Conference Paper
Prothrombin (II) activation is catalyzed by the prothrombinase complex (IIase). Kinetic studies suggested that the principal determinants of substrate or product affinity for IIase are interactions with the extended macromolecular specificity site(s), exosite(s), and not with the active site. Surprisingly, this implies that primary substrate or pro...
Article
Full-text available
The specific action of serine proteinases on protein substrates is a hallmark of blood coagulation and numerous other physiological processes. Enzymic recognition of substrate sequences preceding the scissile bond is considered to contribute dominantly to specificity and function. We have investigated the contribution of active site docking by uniq...
Article
Full-text available
Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we rep...
Article
Full-text available
The coagulation cascade is a complex biochemical network replete with regulatory reactions and cellular contributions. The behavior of the system cannot necessarily be predicted intuitively from the existing state-of-the-art biochemical knowledge of the individual reactions. Knowledge of the
Article
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The binding of recombinant nematode anticoagulant protein c2 (NAPc2) to either factor X or Xa is a requisite step in the pathway for the potent inhibition of VIIa tissue factor. We have used NAPc2 as a tight binding probe of human Xa to investigate protein substrate recognition by the human prothrombinase complex. NAPc2 binds with high affinity (K(...
Article
Full-text available
Kinetic studies support the concept that protein substrate recognition by the prothrombinase complex of coagulation is achieved by interactions at extended macromolecular recognition sites (exosites), distinct from the active site of factor Xa within the complex. We have used this formal kinetic model and a monoclonal antibody directed against Xa (...
Article
Full-text available
The prothrombinase complex, composed of the proteinase, factor Xa, bound to factor Va on membranes, catalyzes thrombin formation by the specific and ordered proteolysis of prothrombin at Arg323-Ile324, followed by cleavage at Arg274-Thr275. We have used a fluorescent derivative of meizothrombin des fragment 1 (mIIaΔF1) as a substrate analog to asse...
Article
The prothrombinase complex (Hase), assembled through reversible interactions between factor Xa and factor Va on membranes, catalyses the conversion of prothrombin to thrombin. Kinetic studies indicate that binding specificity for the protein substrate derives from interactions at exosites within Ilase. The individual contributions of Xa and Va with...
Article
Full-text available
The initiation of coagulation results from the activation of factor X by an enzyme complex (Xase) composed of the trypsin-like serine proteinase, factor VIIa, bound to tissue factor (TF) on phospholipid membranes. We have investigated the basis for the protein substrate specificity of Xase using TF reconstituted into vesicles of phosphatidylcholine...
Article
Full-text available
The proteolytic formation of thrombin is catalyzed by the prothrombinase complex of blood coagulation. The kinetics of prethrombin 2 cleavage was studied to delineate macromolecular substrate structures necessary for recognition at the exosite(s) of prothrombinase. The product, α-thrombin, was a linear competitive inhibitor of prethrombin 2 activat...
Article
Full-text available
Tissue factor (TF) pathway inhibitor (TFPI) regulates factor X activation through the sequential inhibition of factor Xa and the VIIa.TF complex. Factor Xa formation was studied in a purified, reconstituted system, at plasma concentrations of factor X and TFPI, saturating concentrations of factor VIIa, and increasing concentrations of TF reconstitu...
Article
The proteolytic conversion of factor X to Xa by the extrinsic pathway of blood coagulation is catalysed by an enzyme complex (Xase), composed of the serine protease, factor Vila, bound to tissue factor (TF) on phospholipid membranes. We have studied the kinetics of inhibition of the Xase complex using TF reconstituted into vesicles of phosphatidylc...
Article
The prothrombinase complex, composed of factor Xa and factor Va assembled on a membrane surface, catalyzes the proteolytic formation of thrombin during blood coagulation. The molecular basis for the macromolecular substrate specificity of prothrombinase is poorly understood. By kinetic studies of prethrombin 2 cleavage by prothrombinase in the pres...
Article
The prothrombinase complex assembles through reversible interactions between factor Xa, factor Va and acidic phospholipid-containing membranes in the presence of calcium ions. This complex catalyses the conversion of prothrombin to thrombin through two proteolytic steps. We have used prethrombin 2 as a substrate analog for the first cleavage reacti...
Article
The prothrombinase complex assembles through reversible interactions between the protease, factor Xa, the cofactor, factor Va, and acidic phospholipid membranes in the presence of calcium ions. Changes in macromolecular recognition by factor Xa which may result from its interaction with factor Va in the prothrombinase complex have been probed using...
Article
Full-text available
The activation of factor X by the extrinsic coagulation system results from the action of an enzyme complex composed of factor VIIa bound to tissue factor on phospholipid membranes in the presence of calcium ions (extrinsic Xase complex). Proteolysis at the Arg52-Ile53 peptide bond in the heavy chain of factor X leads to the formation of the serine...
Article
The mechanism of binding of blood coagulation cofactor factor Va to acidic-lipid-containing membranes has been addressed. Binding isotherms were generated at room temperature using the change in fluorescence anisotropy of pyrene-labeled bovine factor Va to detect binding to sonicated membrane vesicles containing either bovine brain phosphatidylseri...
Article
Full-text available
The conversion of prothrombin to thrombin requires the cleavage of two peptide bonds and is catalyzed by the prothrombinase complex composed of factors Xa and Va assembled on a membrane surface. Presteady-state kinetic studies of the effects of membranes on the proteolytic reaction were undertaken using model membranes composed of phosphatidylcholi...
Article
Full-text available
The effect of heparin and other glycosaminoglycans on the activation of factor X by the phospholipid membrane-bound human factor IXa-factor VIIIa complex (intrinsic fXase) was studied. Standard heparin inhibited purified intrinsic fXase by 50% at approximately 0.08 unit/ml (0.4 microgram/ml), which is below the normal range of heparin concentration...
Article
The interaction of factor Xa with factor Va on a membrane surface results in the assembly of the prothrombinase complex. The highly specific and multistep interaction between recombinant tick anticoagulant peptide (rTAP) and factor Xa was used to probe perturbations in the macromolecular interaction sites of factor Xa that accompany prothrombinase...
Article
Full-text available
The interaction of factor Xa with factor Va on the membrane surface results in a 3,000-fold increase in the kcat for the activation of prothrombin catalyzed by factor Xa. The reaction between the transition state irreversible inhibitor dansylglutamyl-glycyl-arginyl chloromethyl ketone (DEGRck) and factor Xa was characterized and employed to evaluat...
Article
This chapter outlines the methodology for studying the catalytic properties of the human extrinsic “Xase” complex with respect to its biological substrates, human factors IX and X, and a synthetic fluorescent substrate for factor VIIa. It discusses the reconstitution of tissue factor (TF) apoprotein into membranes and describes the factor IX activa...
Article
The proteolytic activation of prothrombin is catalyzed by an enzyme complex composed of the serine protease factor Xa reversibly associated with the cofactor factor Va on membranes containing negatively charged phospholipid in the presence of calcium ions. Studies of the structure–function relationships in factor Va have been aided by the availabil...
Article
The approaches described in this article have resulted in an increased understanding of the reaction steps involved in the stabilization and assembly of the prothrombinase complex. Because prothrombinase is considered an archetype for some of the other coagulation complexes, the quantitative information derived from these studies (Table I) provides...
Article
Full-text available
Coagulation factor X is activated by the extrinsic Xase complex composed of factor VIIa associated with the integral membrane protein tissue factor. The kinetics of human factor X activation was studied following reconstitution of this reaction system using purified human proteins and synthetic phospholipid vesicles composed of phosphatidylcholine...
Article
Full-text available
The interaction of factor VIIa with tissue factor (TF) results in an increase in the catalytic efficiency for the hydrolysis of several synthetic peptidyl p-nitroanilide substrates by factor VIIa. The binding of human recombinant factor VIIa to recombinant human TF incorporated into vesicles containing phosphatidylcholine (TF/PC) or phosphatidylcho...