H C Whinna

University of North Carolina at Chapel Hill, Chapel Hill, NC, United States

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Publications (32)128.42 Total impact

  • [show abstract] [hide abstract]
    ABSTRACT: Venous thromboembolism (VTE) encompasses both deep vein thrombosis (DVT) and pulmonary embolism (PE), a very serious and potentially lethal complication of DVT [1]. The strongest but most unchangeable risk factor for VTE is age [2]. After the age of 55, the risk of VTE goes up substantially and in those 75 years and older there is a ten-fold increase from the overall population [1,3]. © 2013 International Society on Thrombosis and Haemostasis.
    Journal of Thrombosis and Haemostasis 03/2013; · 6.08 Impact Factor
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    ABSTRACT: Haemostatic effect of compounds for treating haemophilia can be evaluated in various bleeding models in haemophilic mice. However, the doses of factor VIII (FVIII) for normalizing bleeding used in some of these models are reported to be relatively high. The aim of this study was to establish a sensitive venous bleeding model in FVIII knock out (F8-KO) mice, with the ability to detect effect on bleeding at low plasma FVIII concentrations. We studied the effect of two recombinant FVIII products, N8 and Advate(®), after injury to the saphenous vein. We found that F8-KO mice treated with increasing doses of either N8 or Advate(®) showed a dose-dependent increase in the number of clot formations and a reduction in both average and maximum bleeding time, as well as in average blood loss. For both compounds, significant effect was found at doses as low as 5 IU kg(-1) when compared with vehicle-treated F8-KO mice. Normalization of maximum bleeding time was found at doses equal to or above 10 IU kg(-1) N8 or Advate(®), corresponding to plasma concentrations of approximately 10% of the level in wild type mice. The present study adds a new model to the armamentarium of bleeding models used for evaluation of pro-coagulant compounds for treatment of haemophilia. Interestingly, the vena saphena model proved to be sensitive towards FVIII in plasma levels that approach the levels preventing bleeding in haemophilia patients, and may, thus, in particular be valuable for testing of new long-acting variants of e.g. FVIII that are intended for prophylaxis.
    Haemophilia 04/2012; 18(5):782-8. · 3.17 Impact Factor
  • Journal of Thrombosis and Haemostasis 09/2011; 9(9):1862-3. · 6.08 Impact Factor
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    ABSTRACT: PURPOSE. Use of nomograms based on the "heparin correlation value" (HCV)-a value that corresponds to measured activated partial thromboplastin time (aPTT) and that removes the need to revise nomograms in response to a change in the aPTT reagent or coagulometer used-was evaluated as an alternative to traditional aPTT-based anticoagulation nomograms. SUMMARY. Data were collected on patients receiving heparin therapy for selected indications (thrombotic disorders, cardiac conditions, and acute coronary syndromes) during four-month periods before (n = 59) and after (n = 60) implementation of the HCV-based nomograms. The primary endpoints were the rate at which coagulation laboratory measurements were obtained at the appropriate time and the rate of appropriate dosage adjustment in response to reported laboratory values; secondary endpoints included the time to attainment of the first target anticoagulation value. After implementation of HCV-based nomograms, coagulation laboratory measurements were obtained at the appropriate time in (mean ± S.D.) 92.9% ± 12.8% of patients, compared with 80.1% ± 15.5% of patients who received aPTT-based monitoring (p < 0.0001). After implementation of HCV-based monitoring, the rate of correct heparin dosage adjustments was improved (mean ± S.D. 94.7% ± 7.8% versus 89.3% ± 14.0%, p = 0.01), and the time to attainment of the first target anticoagulation value was shorter (mean ± S.D. 16.4 ± 10.6 hours versus 21.5 ± 14.8 hours, p = 0.03). CONCLUSION. The HCV, which relates measured aPTT values to corresponding antifactor Xa concentrations, was substituted for aPTT in heparin nomograms and appeared to be a viable alternative to the aPTT.
    American journal of health-system pharmacy: AJHP: official journal of the American Society of Health-System Pharmacists 05/2011; 68(10):893-8. · 2.10 Impact Factor
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    ABSTRACT: Age-associated cellular senescence is thought to promote vascular dysfunction. p16(INK4a) is a cell cycle inhibitor that promotes senescence and is upregulated during normal aging. In this study, we examine the contribution of p16(INK4a) overexpression to venous thrombosis. Mice overexpressing p16(INK4a) were studied with 4 different vascular injury models: (1) ferric chloride (FeCl(3)) and (2) Rose Bengal to induce saphenous vein thrombus formation; (3) FeCl(3) and vascular ligation to examine thrombus resolution; and (4) lipopolysaccharide administration to initiate inflammation-induced vascular dysfunction. p16(INK4a) transgenic mice had accelerated occlusion times (13.1 ± 0.4 minutes) compared with normal controls (19.7 ± 1.1 minutes) in the FeCl(3) model and 12.7 ± 2.0 and 18.6 ± 1.9 minutes, respectively in the Rose Bengal model. Moreover, overexpression of p16(INK4a) delayed thrombus resolution compared with normal controls. In response to lipopolysaccharide treatment, the p16(INK4a) transgenic mice showed enhanced thrombin generation in plasma-based calibrated automated thrombography assays. Finally, bone marrow transplantation studies suggested increased p16(INK4a) expression in hematopoietic cells contributes to thrombosis, demonstrating a role for p16(INK4a) expression in venous thrombosis. Venous thrombosis is augmented by overexpression of the cellular senescence protein p16(INK4a).
    Arteriosclerosis Thrombosis and Vascular Biology 01/2011; 31(4):827-33. · 6.34 Impact Factor
  • Clinical Chemistry 10/2010; 56(12):1897-9. · 7.15 Impact Factor
  • Transfusion 12/2008; 48(11):2277-8. · 3.53 Impact Factor
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    ABSTRACT: Pulmonary hypertension (PHT) is common in sickle cell disease (SCD). The purpose of this study was to determine whether markers of coagulation activation and inflammation are associated with PHT in SCD. This cross-sectional study was performed using a cohort of patients followed at an adult Sickle Cell Clinic. Pulmonary artery systolic pressure was determined by Doppler echocardiography, and the diagnosis of PHT was defined using age, sex and body mass index-adjusted reference ranges. Clinical laboratory examinations, including hematologic studies and biochemical tests, as well as various measures of coagulation activation, endothelial activation and inflammation, were conducted on SCD subjects and on healthy, race-matched control subjects without SCD. Patients with SCD (n=76) had higher plasma levels of markers of coagulation (thrombin-antithrombin complex, prothrombin fragment F1+2, D-dimer) and endothelial (soluble vascular endothelial cell adhesion molecule, sVCAM) activation compared with control subjects (n=6). SCD patients with PHT (n=26) had significantly higher levels of sVCAM compared with those patients without PHT (n=50). Although PHT patients showed increased plasma measures of coagulation activation, the differences were not statistically significant when compared to those of patients without PHT. HbSS patients with PHT also had a trend towards higher levels of other inflammatory cytokines (interleukins 6, 8 and 10) than HbSS patients without PHT. There was a modest negative correlation between hemoglobin and plasma measures of coagulation and endothelial activation, and modest positive correlations between markers of hemolysis and plasma measures of coagulation and endothelial activation. SCD patients with PHT have higher levels of markers of endothelial activation and other inflammatory markers than patients without PHT. A trend towards an increased level of markers of coagulation activation was observed in SCD patients with PHT compared with that in patients without PHT. Markers of hemolysis are associated with coagulation activation and endothelial dysfunction in SCD patients. Clinical trials of anticoagulants and anti-inflammatory agents are warranted in SCD patients with PHT.
    Haematologica 02/2008; 93(1):20-6. · 5.94 Impact Factor
  • Herbert C Whinna
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    ABSTRACT: There are a myriad of options on where and how to perform thrombosis studies in mice. Models have been developed for systemic thrombosis, larger and smaller vessels of both the arterial and venous systems as well as several different microvascular beds. However, there are important differences between the models and investigators need to be careful and thoughtful when they choose which model to use.
    Thrombosis Research 01/2008; 122 Suppl 1:S64-9. · 3.13 Impact Factor
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    ABSTRACT: Background: Protein C inhibitor (PCI) and antithrombin (AT) are serine protease inhibitors (serpins) that inhibit a wide array of blood coagulation serine proteases including thrombin. Fifty-five Ala-scanned recombinant thrombin mutants were used to determine thrombin residues important for inhibition by PCI with and without the cofactors heparin and thrombomodulin (TM) and compared with the prototypical serpin, AT. Residues around the active site (Tyr50 and Glu202) and the sodium-binding site (Glu229 and Arg233) were required for thrombin inhibition by PCI with and without cofactors. Exosite-2 residues (Arg89, Arg93, Glu94, Arg98, Arg245, Arg248, and Gln251) were critical for heparin-accelerated inhibition of thrombin by PCI. Exosite-1 residues (especially Lys65 and Tyr71) were required for enhanced PCI inhibition of thrombin-TM. Interestingly, we also found that the TM chondroitin sulfate moiety is not required for the approximately 150-fold enhanced rate of thrombin inhibition by PCI. Using the aforementioned thrombin exosite-2 mutants that were essential for heparin-catalyzed PCI-thrombin inhibition reactions we found no change in PCI inhibition rates for thrombin-TM. Collectively, these results show that (i) similar thrombin exosite-2 residues are critical for the heparin-catalyzed inhibition by PCI and AT, (ii) PCI and AT are different in their thrombin-TM inhibition properties, and (iii) PCI has a distinct advantage over AT in the regulation of the activity of thrombin-TM.
    Journal of Thrombosis and Haemostasis 08/2007; 5(7):1486-92. · 6.08 Impact Factor
  • M Hoffman, H C Whinna, D M Monroe
    Journal of Thrombosis and Haemostasis 10/2006; 4(9):2092-3. · 6.08 Impact Factor
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    ABSTRACT: We used 55 Ala-scanned recombinant thrombin molecules to define residues important for inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) in the absence and presence of glycosaminoglycans. We verified the importance of numerous basic residues in anion-binding exosite-1 (exosite-1) and found 4 additional residues, Gln24, Lys65, His66, and Tyr71 (using the thrombin numbering system), that were resistant to HCII inhibition with and without glycosaminoglycans. Inhibition rate constants for these exosite-1 (Q24A, K65A, H66A, Y71A) thrombin mutants (0.02-0.38 x 10(8) m(-1) min(-1) for HCII-heparin when compared with 2.36 x 10(8) m(-1) min(-1) with wild-type thrombin and 0.03-0.53 x 10(8) m(-1) min(-1) for HCII-dermatan sulfate when compared with 5.23 x 10(8) m(-1) min(-1) with wild-type thrombin) confirmed that the structural integrity of thrombin exosite-1 is critical for optimal HCII-thrombin interactions in the presence of glycosaminoglycans. However, our results are also consistent for HCII-glycosaminoglycan-thrombin ternary complex formation. Ten residues surrounding the active site of thrombin were implicated in HCII interactions. Four mutants (Asp51, Lys52, Lys145/Thr147/Trp148, Asp234) showed normal increased rates of inhibition by HCII-glycosaminoglycans, whereas four mutants (Trp50, Glu202, Glu229, Arg233) remained resistant to inhibition by HCII with glycosaminoglycans. Using 11 exosite-2 thrombin mutants with 20 different mutated residues, we saw no major perturbations of HCII-glycosaminoglycan inhibition reactions. Collectively, our results support a "double bridge" mechanism for HCII inhibition of thrombin in the presence of glycosaminoglycans, which relies in part on ternary complex formation but is primarily dominated by an allosteric process involving contact of the "hirudin-like" domain of HCII with thrombin exosite-1.
    Journal of Biological Chemistry 11/2004; 279(41):43237-44. · 4.65 Impact Factor
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    ABSTRACT: We studied the RNA aptamer Toggle-25/thrombin interaction during inhibition by antithrombin (AT), heparin cofactor II (HCII) and protein C inhibitor (PCI). Thrombin inhibition was reduced 3-fold by Toggle-25 for AT and HCII, but it was slightly enhanced for PCI. In the presence of glycosaminoglycans, AT and PCI had significantly reduced thrombin inhibition with Toggle-25, but it was only reduced 3-fold for HCII. This suggested that the primary effect of aptamer binding was through the heparin-binding site of thrombin, anion-binding exosite-2 (exosite-2). We localized the Toggle-25 binding site to Arg 98, Glu 169, Lys 174, Asp 175, Arg 245, and Lys 248 of exosite-2. We conclude that a RNA aptamer to thrombin exosite-2 might provide an effective clinical reagent to control heparin's anticoagulant action.
    FEBS Letters 07/2004; 568(1-3):10-4. · 3.58 Impact Factor
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    ABSTRACT: Two patients, treated with enoxaparin and eptifibatide, developed significant guide catheter-associated thrombus while undergoing intravascular ultrasound (IVUS). Using an ex vivo assay, we found that activation of a multielement IVUS catheter resulted in a decrease in anti-Xa activity and a decrease in clot formation time. This effect occurred rapidly and repeatedly after activation of the IVUS catheter.
    The American Journal of Cardiology 07/2004; 93(11):1453-4, A12. · 3.21 Impact Factor
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    ABSTRACT: Factor (F)Xa has 11 gamma-carboxylated glutamic acid (Gla) residues that are involved in calcium-dependent membrane binding. The serpin antithrombin (AT) is an important physiological regulator of FXa activity in an inhibition reaction that is enhanced by heparin. Recently, Rezaie showed that calcium further enhanced the heparin-catalyzed AT inhibition of FXa by promoting 'ternary complex' formation, and these results showed a role for the gamma-carboxyl-glutamate (Gla)-domain of FXa. In this study, we used recombinant FXa mutants to assess the role of individual Gla residues in augmenting or antagonizing the AT-heparin inhibition reaction in the presence of calcium. In the absence of heparin, AT inhibition of plasma and the recombinant FXas were essentially equivalent. Similar to plasma-derived FXa, calcium increased about 3-fold the inhibition rate of wild-type recombinant FXa by AT-heparin over that in the presence of EDTA. Interestingly, three different effects were found with the recombinant FXa Gla-mutants for AT-heparin inhibition: (i) Gla-->Asp 14 and 29 were enhanced without calcium; (ii) Gla-->Asp 16 and 26 were not enhanced by calcium; and (iii) Gla-->Asp 19 was essentially the same as wild-type recombinant FXa. These results support a theory that mutating individual Gla residues in FXa alters the calcium-induced conformational changes in the Gla region and affects the antithrombin-heparin inhibition reaction.
    Journal of Thrombosis and Haemostasis 07/2004; 2(7):1127-34. · 6.08 Impact Factor
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    ABSTRACT: Protein C inhibitor (PCI) inhibits multiple plasma serine proteases. To determine which residues contribute to its specificity of inhibition, 19 mutations in the reactive site loop of PCI (from Thr352 to Arg357) were generated and assayed with thrombin, activated protein C (APC), and factor Xa. To identify the intermolecular interactions responsible for these kinetics, a molecular model of PCI was generated using alpha 1-protease inhibitor and ovalbumin as templates. This model of PCI was docked with thrombin, followed by extensive energy minimization, to determine a lowest energy complex. The resulting docked complex was used as a template to form molecular models of PCI-APC and PCI-factor Xa complexes. The best inhibitors of thrombin contained Pro or Gly at the P2 position in place of Phe353, with 2- and 7-fold increases in activity, respectively. These substitutions reduced steric interactions with the 60-insertion loop unique to thrombin. The best inhibitors of APC and factor Xa contained Arg at the P3 position in place of Thr352, with 2- and 5-fold increases in inhibition rates, respectively. The molecular model predicts that Arg in this position could form a salt bridge with Glu217 of each protease. Changing Arg357 at the P3' position had little effect on protease inhibition, consistent with the observation in the model that this residue points toward the body of PCI, forming a salt bridge with Glu220. Given its broad specificity of inhibition, PCI has proven very useful in understanding the nature of serpin-protease interactions using multiple mutations in a serpin assayed with multiple proteases.
    Biochemistry 04/2002; 34(40). · 3.38 Impact Factor
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    Susannah J Bauman, Herbert C Whinna, Frank C Church
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    ABSTRACT: Serpins are a class of proteins involved in the regulation of serine and other types of proteases. In humans, the majority of serpins regulate the functions of proteases involved in the body's response to injury. This includes roles in coagulation, fibrinolysis, inflammation, wound healing, and tissue repair. Serpins have been implicated in various animal and human pathologies by the loss of a functional serpin gene through deletion or mutation, which results in a defect in functional protein. Examples of sestorically called antithrombin III) are first described. Then, protocols to determine the second-order rate constant of AT inhibition of thrombin in the absence and presence of heparin are presented. Also provided is a partial list of other serpins and their purification methods.
    Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 03/2002; Chapter 21:Unit 21.7.
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    ABSTRACT: 'Thrombin aptamers' are based on the 15-nucleotide consensus sequence of d(GGTTGGTGTGGTTGG) that binds specifically to thrombin's anion-binding exosite-I. The effect of aptamer-thrombin interactions during inhibition by the serine protease inhibitor (serpin) heparin cofactor II (HCII) and antithrombin (AT) has not been described. Thrombin inhibition by HCII without glycosaminoglycan was decreased approximately two-fold by the aptamer. In contrast, the aptamer dramatically reduced thrombin inhibition by >200-fold and 30-fold for HCII-heparin and HCII-dermatan sulfate, respectively. The aptamer had essentially no effect on thrombin inhibition by AT with or without heparin. These results add to our understanding of thrombin aptamer activity for potential clinical application, and they further demonstrate the importance of thrombin exosite-I during inhibition by HCII-glycosaminoglycans.
    FEBS Letters 11/2000; 484(2):87-91. · 3.58 Impact Factor
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    ABSTRACT: Asparaginase (ASNase) is a widely used and successful agent against childhood acute lymphoblastic leukemia (ALL). Asparaginase cleaves asparagine (Asn) to give aspartic acid and ammonia, thereby depleting free Asn in the blood. However, treatment with ASNase has been implicated in significant reduction of plasma levels of the coagulation serine protease inhibitor (serpin) antithrombin III (AT3), predisposing patients to thromboembolic complications. Our investigation was designed to delineate the biochemical mechanism of AT3 depletion that can occur in the plasma of ALL patients undergoing ASNase therapy. SDS-PAGE showed no cleavage of purified AT3 following treatment with ASNase. Furthermore, purified AT3 treated with ASNase demonstrated no decrease in inhibitory activity. Human plasma and whole blood treated with approximate therapeutic concentrations of ASNase showed no loss of AT3 activity as detected by a plasma-based factor Xa inhibition assay. Treatment of a confluent monolayer of HepG2 (hepatocarcinoma) cells with ASNase showed no gross loss in AT3 message levels detected by rtPCR. However, a decrease of cell viability was observed in cultures treated with ASNase. Interestingly, medium from HepG2 cells treated with ASNase showed a marked decrease in secretion of AT3 and another serpin, heparin cofactor II. Collectively, these data show that ASNase has no direct effect on AT3 in blood or plasma, but that ASNase may affect plasma levels of AT3 by interfering with translation and/or secretion of the protein in liver cells.
    Leukemia Research 08/2000; 24(7):559-65. · 2.76 Impact Factor
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    ABSTRACT: Site-directed mutagenesis was used to investigate the role of basic residues in the thrombin anion-binding exosite-I during formation of thrombin-antithrombin III (ATIII), thrombin-protease nexin 1 (PN1), and thrombin-heparin cofactor II (HCII) inhibitor complexes, in the absence and presence of glycosaminoglycans. In the absence of glycosaminoglycan, association rate constant (kon) values for the inhibition of the mutant thrombins (R35Q, K36Q, R67Q, R73Q, R75Q, R77(a)Q, K81Q, K109Q, K110Q, and K149(e)Q) by ATIII and PN1 were similar to wild-type recombinant thrombin (rIIa), whereas kon values were decreased 2-3-fold for HCII against the majority of the exosite-I mutants. The exosite-I mutants did not have a significant effect on heparin-accelerated inhibition by ATIII with maximal kon values similar to rIIa. A small effect was seen for PN1/heparin inhibition of the exosite-I mutants R35Q, R67Q, R73Q, R75Q, and R77(a)Q, where kon values were decreased 2-4-fold, compared with rIIa. For HCII/heparin, kon values for inhibition of the exosite-I mutants (except R67Q, R73Q, and K149(e)Q) were 2-3-fold lower than rIIa. Larger decreases in kon values for HCII/heparin were found for R67Q and R73Q thrombins with 441- and 14-fold decreases, respectively, whereas K149(e)Q was unchanged. For HCII/dermatan sulfate, R67Q and R73Q had kon values reduced 720- and 48-fold, respectively, whereas the remaining mutants were decreased 3-7-fold relative to rIIa. The results suggest that ATIII has no major interaction with exosite-I of thrombin with or without heparin. PN1 bound to heparin uses exosite-I to some extent, possibly by utilizing the positive electrostatic field of exosite-I to enhance orientation and thrombin complex formation. The larger effects of the thrombin exosite-I mutants for HCII inhibition with heparin and dermatan sulfate indicate its need for exosite-I, presumably through contact of the "hirudin-like" domain of HCII with exosite-I of thrombin.
    Journal of Biological Chemistry 12/1998; 273(47):31203-8. · 4.65 Impact Factor