von Willebrand factor stimulates thrombin-induced exposure of procoagulant phospholipids on the surface of fibrin-adherent platelets

Summary. Studies from our laboratory have demonstrated that von Willebrand factor (VWF) stimulates thrombin generation in platelet‐rich plasma. The precise role of VWF and fibrin in this reaction, however, remained to be clarified. In the present study we utilized thrombin‐free planar fibrin layers and washed platelets to examine the relationship between platelet–fibrin interaction and exposure of coagulation‐stimulating phosphatidylserine (PS) under conditions of low and high shear stress. Our study confirms that platelet adhesion to fibrin at a shear rate of 1000 s^?1 requires fibrin‐bound VWF. The cytosolic calcium concentration ([Ca²⁺]_i) of stationary platelets was not elevated and PS exposing platelets were virtually absent (2 ± 2%). However, thrombin activation resulted in a marked increase in the number of PS exposing platelets (up to 85 ± 14%) along with a transient elevation in [Ca²⁺]_i from 0.05 µmol L^?1 up to 1.1 ± 0.2 µmol L^?1. Platelet adhesion to fibrin at a shear rate of 50 s^?1 is mediated by thrombin but not by fibrin‐bound VWF. The [Ca²⁺]_i of these thrombin‐activated platelets was elevated (0.2 ± 0.1 µmol L^?1), but only a minority of the platelets (11 ± 8%) exposed PS. The essential role of VWF in this thrombin‐induced procoagulant response became apparent from low shear rate perfusion studies over fibrin that was incubated with VWF and botrocetin. After treatment with thrombin, the majority of the adherent platelets (57 ± 23%) exposed PS and had peak values of [Ca²⁺]_i of about 0.6 µmol L^?1. Taken together, these results demonstrate that thrombin‐induced exposure of PS and high calcium response on fibrin‐adherent platelets depends on shear‐ or botrocetin‐induced VWF–platelet interaction.     

Intrinsic stability and functional properties of disulfide bond-stabilized coagulation factor VIIIa variants

BACKGROUND: The utility of purified coagulation factor (F)VIII for treatment of hemophilia A is limited in part by its instability following activation by thrombin, which is caused by spontaneous dissociation of the A2 domain from the activated FVIII (FVIIIa) heterotrimer. To prevent this A2 domain dissociation in FVIIIa, we previously engineered a cysteine pair (C664-C1826) in recombinant FVIII that formed a disulfide bond cross-linking the A2 domain in the heavy chain to the A3 domain in the light chain. This engineered disulfide bond resulted in a more stable FVIIIa. AIMS: Here, we characterize the functional parameters of C664-C1828 FVIII and of a new disulfide bond-stabilized FVIII (C662-C1828 FVIII). METHODS: In order to assess whether these FVIII variants might be good candidates for a new therapeutic agent to treat hemophilia A, we investigated a variety of functional parameters that might affect the in vivo properties of the variants, including half-life of disulfide bond-stabilized FVIII and FVIIIa and the potency of these FVIIIa molecules in the FXase complex. RESULTS: Both disulfide bond-stabilized variants had improved affinity for von Willebrand factor (VWF). In studies of FX activation by purified FIXa and FVIIIa, C662-C1828 FVIIIa had normal activity while C664-C1826 FVIIIa had reduced activity. Both C664-C1826 FVIIIa and C662-C1828 FVIIIa were inactivated by activated protein C (APC) but the rates of inactivation were different. CONCLUSION: Overall, the specific location of the disulfide bridge between the A2 and A3 domains appears to affect functional properties of FVIIIa. In summary, introduction of engineered interdomain disulfides results in FVIIIa variants that resist spontaneous loss of activity while retaining susceptibility to APC proteolytic inactivation and maintaining VWF binding.     

A3 domain residue Glu1829 contributes to A2 subunit retention in factor VIIIa

BACKGROUND: Factor VIII (FVIII) is activated by thrombin to the labile FVIIIa, a heterotrimer of A1, A2 and A3C1C2 subunits, which serves as a cofactor for FIXa. A primary reason for the instability of FVIIIa is the tendency for the A2 subunit to dissociate from FVIIIa leading to an inactive cofactor and consequent loss of FXase activity. OBJECTIVE: Based on our finding of low-specific activity and a fast decay rate for a FVIII point mutation of Glu1829 to Ala (E1829A), we examined whether residue Glu1829 in the A3 subunit is important for A2 subunit retention. RESULTS: The rate of activity decay of E1829A was approximately fourteen fold faster than wild-type (wt) FVIIIa and this rate was reduced in the presence of added A2 subunit. Specific activity values for E1829A measured by one-stage and two-stage assays were approximately 14% and approximately 11%, respectively, compared with wt FVIII. Binding affinity for the A1 subunit to E1829A-A3C1C2 was comparable to wt A3C1C2 (K(d) = 20.1 +/- 3.4 nM for E1829A, 15.3 +/- 3.7 nM for wt), whereas A2 subunit affinity for the A1/A3C1C2 dimer forms was reduced by approximately 3.6-fold as a result of the mutation (K(d) = 526 +/- 107 nM for E1829A, 144 +/- 21 nM for wt). CONCLUSION: As modeling data suggest that Glu1829 is located at the A2-A3 domain interface these results are consistent with Glu1829 contributing to the interactions involved with A2 subunit retention in FVIIIa.     

An engineered interdomain disulfide bond stabilizes human blood coagulation factor VIIIa

Summary.  The blood coagulation disorder, hemophilia A, is caused by deficiency of coagulation factor (F)VIII. Hemophilia A is now treated by infusions of pure FVIII, but the activity of FVIII is limited because it is unstable following activation by thrombin. This instability of activated FVIII is the result of dissociation of the A2 subunit. To obtain increased stability in FVIIIa, a disulfide bond between the A2 domain and the A3 domain, preventing A2 subunit dissociation, has been engineered. Structural analysis of the FVIII A domain homology model allowed us to identify residues 664 and 1826 as a potential disulfide bond pair. A FVIII mutant containing Cys664 and Cys1826 was produced and purified (C664-C1826 FVIII). Immunoblotting showed that a disulfide bond did form to link covalently the A2 and the A3 domains. Following activation of the recombinant C664-C1826 FVIII by thrombin, the mutant FVIIIa had increased stability and retained more than 90% of its clotting activity at a time at which wild-type FVIIIa lost more than 90% of its activity. This remarkably stable C664-C1826 FVIIIa provides a unique approach for studies of the cofactor activity of FVIIIa and also for new, improved therapy for hemophilia A.     

Co-localization of von Willebrand factor with platelet thrombi, tissue factor and platelets with fibrin, and consistent presence of inflammatory cells in coronary thrombi obtained by an aspiration device from patients with acute myocardial infarction

BACKGROUND: Detailed histochemical analysis of coronary thrombi obtained freshly from acute phase of myocardial infarction patients may provide information necessary to understand the mechanism of coronary occlusive thrombus formation. METHODS AND RESULTS: Coronary thrombi causing myocardial infarction were obtained from 10 consecutive patients of myocardial infarction in the acute phase, using a newly developed aspiration catheter. All the fixed specimens of coronary thrombi, by hematoxylin and eosin staining, were found to contain three major constituents, namely, platelets, densely packed fibrin and inflammatory cells, including polymorphonuclear and mononuclear cells, although their distribution in each specimen is totally heterogeneous. Immunohistochemical staining revealed the prominent presence of von Willebrand factor (VWF) at the sites of platelet accumulation, presence of tissue factor and platelets at the sites of deposition of fibrin fibrils. It also revealed the presence of CD16-, CD45- and CD34-positive cells, yet the functional roles of these cells have still to be elucidated. There are weak positive correlation between the number of inflammatory cells involved in the unit area of coronary thrombi specimen and the time of collection of the specimens after the onset of chest pain. CONCLUSIONS: In spite of various limitations, our results contain information suggesting the possible role of VWF in platelet-thrombus formation, possible important role played by tissue factor and activated platelets in the formation of fibrin fibrils, and the positive relationship between inflammatory cells migration and the formation of occlusive thrombi in human coronary arteries.     

Clustered basic residues within segment 484–510 of the factor VIIIa A2 subunit contribute to the catalytic efficiency for factor Xa generation

Summary.  Residues 484–510 of factor (F)VIIIa A2 subunit comprise a prominent epitope for inhibitor antibodies, suggesting that this region is critical for cofactor function. To address the role of this region in catalysis, FVIIIa forms were evaluated following conversion of conserved charged residues to Ala, either in clusters or individually. The two cluster mutants, Lys496Ala/Lys499Ala/Asp500Ala and Glu507Ala/Lys510Ala, were indistinguishable from wild type. The mutation Arg489Ala/Arg490Ala/Lys493Ala (489-3A) possessed near-normal affinity for FIXa and showed no effect on the Km for FX, but exhibited ∼ 3-fold and ∼ 30-fold reduced kcat values for FXase in the presence and absence of surface, respectively. However, the single-site mutants Arg489Ala, Arg490Ala and Lys493Ala exhibited affinity and kcat values similar to wild type. Furthermore, the 489-3A mutant showed a marked reduction in the positive electrostatic potential within this region of A2, consistent with the hypothesis that the cumulative basic charge in this region of A2 subunit modulates cofactor function.     

Disulfide bond-stabilized factor VIII has prolonged factor VIIIa activity and improved potency in whole blood clotting assays

BACKGROUND: Genetically engineered disulfide bonds in B-domain-deleted factor (F) VIII variants (C662-C1828 FVIII and C664-C1826 FVIII) improve FVIIIa stability by blocking A2 domain dissociation because the new disulfide covalently links the A2 and A3 domains in FVIIIa. AIM: The aim of this study was to assess the hypothesis that these variants have physiologically relevant properties because of prolonged thrombin generation and improved clot formation in whole blood. METHODS: Clot-formation properties in whole blood were measured in thromboelastogram assays. The thrombin generation capabilities of the wild-type (WT) FVIII and FVIII variants were determined, and half-lives of FVIIIa variants were determined in fresh whole blood serum. RESULTS: Thromboelastogram assays were performed with fresh, severe hemophilia whole blood reconstituted with variant and WT FVIII. The two disulfide bond-stabilized FVIII variants and WT FVIII had comparable clotting times at all studied concentrations. However, when compared with WT FVIII at low concentrations, the two FVIII variants required only 10% as much FVIII to achieve comparable clot-formation rates, clot-formation times and clot firmness values. The differences between WT and FVIII variants were quite pronounced at low FVIII concentrations. Measurement of the endogenous thrombin potential in FVIII-deficient plasma supplemented with these FVIII variants confirmed that the disulfide bond-stabilized variants supported high levels of thrombin generation at lower concentrations than did WT FVIII. During the course of clot generation in whole blood, the disulfide bond-stabilized FVIIIa variants had approximately 5-fold increased half-lives relative to WT FVIIIa. CONCLUSION: C662-C1828 FVIII and C664-C1826 FVIII have physiologically relevant superior clot-forming properties in a whole blood environment, most likely due to the increased half-life of these FVIIIa variants in whole blood.     

Laminin promotes coagulation and thrombus formation in a factor XII‐dependent manner

Summary. Background: Laminin is the most abundant non‐collagenous protein in the basement membrane. Recent studies have shown that laminin supports platelet adhesion, activation and aggregation under flow conditions, highlighting a possible role for laminin in hemostasis. Objective: To investigate the ability of laminin to promote coagulation and support thrombus formation under shear. Results and methods: Soluble laminin accelerated factor (F) XII activation in a purified system, and shortened the clotting time of recalcified plasma in a FXI‐ and FXII‐dependent manner. Laminin promoted phosphatidylserine exposure on platelets and supported platelet adhesion and fibrin formation in recalcified blood under shear flow conditions. Fibrin formation in laminin‐coated capillaries was abrogated by an antibody that interferes with FXI activation by activated FXII, or an antibody that blocks activated FXI activation of FIX. Conclusion: This study identifies a role for laminin in the initiation of coagulation and the formation of platelet‐rich thrombi under shear conditions in a FXII‐dependent manner.     

Factor H binds to washed human platelets.

BACKGROUND: Factor H regulates the alternative pathway of complement. The protein has three heparin-binding sites, is synthesized primarily in the liver and copurifies from platelets with thrombospondin-1. Factor H mutations at the C-terminus are associated with atypical hemolytic uremic syndrome, a condition in which platelets are consumed. Objectives The aim of this study was to investigate if factor H interacts with platelets. METHODS: Binding of factor H, recombinant C- or N-terminus constructs and a C-terminus mutant to washed (plasma and complement-free) platelets was analyzed by flow cytometry. Binding of factor H and constructs to thrombospondin-1 was measured by surface plasmon resonance. RESULTS: Factor H bound to platelets in a dose-dependent manner. The major binding site was localized to the C-terminus. The interaction was partially blocked by heparin. Inhibition with anti-GPIIb/IIIa, or with fibrinogen, suggested that the platelet GPIIb/IIIa receptor is involved in factor H binding. Factor H binds to thrombospondin-1. Addition of thrombospondin-1 increased factor H binding to platelets. Factor H mutated at the C-terminus also bound to platelets, albeit to a significantly lesser degree. CONCLUSIONS: This study reports a novel property of factor H, i.e. binding to platelets, either directly via the GPIIb/IIIa receptor or indirectly via thrombospondin-1, in the absence of complement. Binding to platelets was mostly mediated by the C-terminal region of factor H and factor H mutated at the C-terminus exhibited reduced binding.     

Binding studies of the enzyme (factor IXa) with the cofactor (factor VIIIa) in the assembly of factor-X activating complex on the activated platelet surface

Summary.  Activated platelet membranes expose binding sites for the enzyme factor (F)IXa, the substrate (FX) and the cofactor (FVIIIa) that colocalize to assemble the FX-activating complex and promote optimal rates of FX activation. To determine the stoichiometry and affinity of binding to activated platelets, coordinate, equilibrium binding studies with enzyme (¹²⁵I-FIXa) and cofactor (¹³¹I-FVIII or ¹³¹I-FVIIIa) were carried out in the presence of saturating concentrations of substrate (FX). Results of these studies indicate that in the presence of FX (1.5 µ m ), the enzyme (active-site-inhibited Glu-Gly-Arg-FIXa, EGR-FIXa) and procofactor (FVIII) bind to an equal number (approximately 700 sites/platelet) of receptors whereas the active cofactor (FVIIIa) binds an additional approximately 500 high-affinity FVIIIa binding sites per platelet (K_d approximately 0.8 n m ). With excess zymogen (FIX) to block shared FIX/FIXa-binding sites, the stoichiometry of ¹²⁵I-FIXa and ¹³¹I-FVIIIa binding was 1 : 4. These FIXa/FVIIIa binding studies together with previously reported evidence of the coordinate binding of FVIIIa and FX to equivalent numbers of binding sites on activated platelets provide strong evidence to support the conclusion that FVIIIa comprises the receptor that presents FX to FIXa for efficient catalysis on the activated platelet membrane.     

Abciximab inhibits procoagulant activity but not the release reaction upon collagen- or clot-adherent platelets

Summary.  In addition to inhibiting platelet (plt) aggregation abciximab, a glycoprotein (GP) IIb/IIIa antagonist, reduces coagulation in blood or platelet-rich plasma. We assessed the effects of abciximab (10 µg mL⁻¹) on adhesion-dependent procoagulant activity (PCA) of plt upon: (i) collagen to model initial adhesion; or (ii) plasma clot or fibrin to model a preformed thrombus with retained thrombin activity. In a two-stage assay gel-filtered plt (GFP) first adhered on collagen, plasma clot, or fibrin, and plt activation was traced with platelet factor 4 (PF 4) release. Second, PCA was measured on adherent plt (i) by soluble prothrombin fragments (F1 + 2); and (ii) chromogenically by adding defibrinated plasma and thromboplastin. Abciximab inhibited aggregation upon collagen-adherent plt both in the absence and presence of plasma. In contrast, without plasma abciximab enhanced plt deposition to fibrin surfaces depending on thrombin generation and fibrin polymerization. However, abciximab reduced PCA and generation of F1 + 2 on adherent plt surface-independently by 35%, whereas PF 4 release persisted. Also, a GP Ib inhibitor, mAb SZ2, attenuated PCA by 40% alone, and by 65% together with abciximab, leaving 35% of PCA unaltered. Abciximab decreased generation of new thrombin on both collagen- and clot-adherent plt. However, abciximab did not inhibit α-granule release, suggesting distinct pathways for PCA and release reaction. Deposition of isolated plt on clots in the presence of abciximab was dependent on thrombin and polymerizing plt-derived fibrin(ogen). Due to local consumption of natural anticoagulants adjacent to a preformed thrombus the antithrombotic effect of abciximab benefits from additional inhibition of thrombin.     

Preferential localization of recombinant factor VIIa to platelets activated with a combination of thrombin and a glycoprotein VI receptor agonist

BACKGROUND: Activation of platelets with a combination of collagen and thrombin generates a subpopulation of highly procoagulant 'coated' platelets characterized by high surface expression of fibrinogen and other procoagulant proteins. OBJECTIVES: To analyze the interaction of recombinant factor VIIa (rFVIIa) with coated platelets. METHODS AND RESULTS: rFVIIa localized to the coated platelets in flow cytometry experiments, while minimal rFVIIa was found on platelets activated with adenosine diphosphate, thrombin or via glycoprotein VI individually, and essentially no rFVIIa was found on non-stimulated platelets. Removal of the gamma-carboxyglutamic acid (Gla) domain of rFVIIa, and addition of EDTA, annexin V or excess prothrombin inhibited rFVIIa localization to the coated platelets, indicating that the interaction was mediated by the calcium-dependent conformation of the Gla domain and platelet exposure of negatively charged phospholipids. A reduced level of platelet fibrinogen exposure was observed at hemophilia A-like conditions in a model system of cell-based coagulation, indicating that coated platelet formation in hemophilia may be diminished. Addition of rFVIIa dose-dependently enhanced thrombin generation and partly restored platelet fibrinogen exposure. CONCLUSIONS: The data suggest that rFVIIa localized preferentially on platelets activated with dual agonists, thereby ensuring enhanced thrombin generation localized at the site of injury where both collagen and tissue factor are exposed, the latter ensuring the formation of thrombin necessary for coated platelet formation.     

Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.     

Extra-hepatic factor VIII expression in porcine fulminant hepatic failure

In humans, fulminant hepatic failure (FHF) is frequently associated with increased factor VIII (FVIII) levels, despite widespread liver cell death. The mechanisms leading to increased FVIII levels and cellular sites of this enhanced FVIII production are poorly understood. We studied the effect of total hepatectomy in pigs, a large-animal model of FHF, on the expression of plasma and tissue FVIII during 24-hour follow-up. Tissue FVIII expression was determined before and 24 h after hepatectomy, both at the mRNA level and immunohistochemically. The expression of plasma and tissue von Willebrand factor (VWF), the natural stabilizing carrier protein of FVIII, was also measured. Total hepatectomy elicited a gradual and sustained twofold elevation of circulating FVIII, whereas FVIII mRNA levels in various organs did not increase after hepatectomy. The half-life of FVIII increased from 7.7 to 10.3 h and VWF levels were also elevated in anhepatic pigs. The increase in the half-life of FVIII and increased levels of VWF were not sufficient to explain the rise in plasma FVIII levels. At the protein level, prominent changes in the cellular distribution of FVIII were seen in spleen and kidney. These observations suggest that in this model of FHF the lack of hepatic FVIII synthesis is adequately compensated by other organs, notably spleen and kidneys.     

The assembly of the factor X-activating complex on activated human platelets

Summary.  Platelet membranes provide procoagulant surfaces for the assembly and expression of the factor X-activating complex and promote the proteolytic activation and assembly of the prothrombinase complex resulting in normal hemostasis. Recent studies from our laboratory and others indicate that platelets possess specific, high-affinity, saturable, receptors for factors XI, XIa, IX, IXa, X, VIII, VIIIa, V, Va and Xa, prothrombin, and thrombin. Studies described in this review support the hypothesis that the factor X-activating complex on the platelet surface consists of three receptors (for the enzyme, factor IXa; the substrate, factor X; and the cofactor, factor VIIIa), the colocalization of which results in a 24 million-fold acceleration of the rate of factor X activation. Whether the procoagulant surface of platelets is defined exclusively by procoagulant phospholipids, or whether specific protein receptors exist for the coagulant factors and proteases, is currently unresolved. The interaction between coagulation proteins and platelets is critical to the maintenance of normal hemostasis and is pathogenetically important in human disease.     

Mild hemophilia A with factor VIII assay discrepancy: using thrombin generation assay to assess the bleeding phenotype

Summary.  Introduction:  In some patients with mild hemophilia A, there are discrepancies between 1-stage (1-st) and 2-stage (2-st) factor VIII (FVIII) clotting assays, and also chromogenic assays for FVIII activity (FVIII:C). We examined whether thrombography could provide a better evaluation of the hemostatic status of these patients. Methods:  Two families with such discrepancies and markedly contrasting clinical histories were studied. Family X had no serious bleedings, in contrast to family Y. Sixty-one moderate/mild hemophiliacs without discrepancy and 15 healthy subjects served as controls. Calibrated automated thrombography was performed with platelet-rich plasma after one freeze-thawing cycle and low tissue factor concentration. Results:  The chromogenic FVIII:C levels were higher (0.90 ± 0.15 and 0.47 ± 0.13 IU mL⁻¹) than the 1-st clotting ones (0.14 ± 0.05 and 0.10 ± 0.05 IU mL⁻¹) in family X and Y, respectively ( P  < 0.001). Mean endogenous thrombin potential (ETP) was 1579 ± 359 n m  min⁻¹ and 1060 ± 450 for healthy controls and hemophilic controls, respectively. For members of family X, the ETP values were 1188, 1317 and 2277 n m  min⁻¹, whereas for those of family Y they ranged from 447 to 1122 n m  min⁻¹. Two novel missense point mutations were evidenced: p.Ile369Thr in family X and p.Phe2127Ser in family Y. In family X, we postulate that the mutation is responsible for a delayed but non-deleterious FVIII activation. Conclusions:  Our results suggest that the hemostatic phenotype assessed by thrombography may be clinically relevant in moderate/mild hemophilic patients with discrepant FVIII:C results.