Platelet binding and activity of recombinant factor VIIa

Recombinant FVIIa was developed for the purpose of treating hemophiliacs with antibody inhibitors. It was initially assumed to act by enhancing factor X activation by a tissue factor-dependent mechanism. However, the very high levels of FVIIa required for hemostatic effect in vivo seemed inconsistent with this mechanism. After many years of debate, in now appears that platelet surface binding and activity play an important role in the efficacy of FVIIa as a bypassing agent in hemophilia. Platelet binding was initially suggested to be mediated by binding to anionic phospholipid exposed on platelet surfaces upon activation. It now appears that the glycoprotein Ib/IX/V complex also plays a role in FVIIa binding to platelets. However, the characteristics of FVIIa binding to GPIb/IX/V to not seem to fully explain platelet localization of FVIIa to platelets. Thus, there are still unanswered questions in fully understanding the mechanism of hemostatic action of recombinant FVIIa.     

The effect of temperature and pH on the activity of factor VIIa: implications for the efficacy of high-dose factor VIIa in hypothermic and acidotic patients

BACKGROUND: Recombinant coagulation factor VIIa (FVIIa) is approved for treating hemophiliacs with inhibitors. High-dose FVIIa has also been used off-label to manage hemorrhage in trauma and surgical patients, many of whom also develop hypothermia and acidosis. METHODS: We examined the activity of FVIIa on phospholipid vesicles in the presence and absence of tissue factor (TF) and on platelets as a function of temperature and pH. RESULTS: FVIIa activity on phospholipids and platelets was not reduced at 33 degrees C compared with 37 degrees C. The activity of FVIIa/TF was reduced by 20% at 33 degrees C compared with 37 degrees C. A pH decrease from 7.4 to 7.0 reduced the activity of FVIIa by over 90% and FVIIa/TF by over 60%. CONCLUSION: FVIIa should be effective in enhancing hemostasis in hypothermic patients. However, because the activity of FVIIa is so dramatically affected by pH, its efficacy may be reduced in acidotic patients.     

Elevated prothrombin results in clots with an altered fiber structure: a possible mechanism of the increased thrombotic risk

Individuals with elevated prothrombin levels are at increased risk of venous thrombosis. To understand the mechanism behind this observation, we studied the effect of prothrombin concentration on thrombin generation and fibrin clot structure. The pattern of thrombin generation was directly related to the prothrombin level at all concentrations tested. From 0% to 300% of normal plasma levels of prothrombin, increasing the prothrombin concentration increased the initial rate, peak, and total amount of thrombin generated. Importantly, fibrin clot structure was also affected by the prothrombin concentration. Fibrin clots made from prothrombin concentrations less than 10% of plasma levels were weak and poorly formed. Fibrin clots made at 10% to 100% of plasma levels of prothrombin had similar fiber structures (mass-to-length ratio; mu). However, the fiber mass-to-length ratio decreased with increasing prothrombin levels more than 100% of plasma levels, in a dose-dependent manner. These results suggest that increased levels of prothrombin alter thrombin generation and clot structure. Specifically, elevated prothrombin levels produce clots with reduced fibrin mass-to-length ratios compared with normal clots. We hypothesize that this alteration in fibrin clot structure is an important determinant of the risk of thrombosis.     

Hypothesis: hyperhomocysteinemia is an indicator of oxidant stress

Elevated plasma homocysteine levels are associated with an increased risk of atherosclerosis and thrombosis, as well as a variety of other pathologies such as birth defects, Alzheimer’s disease and other dementias, osteoporosis, diabetes and renal disease. Homocysteine metabolism is catalyzed by a number of enzymes that require B-vitamins as cofactors, and homocysteine levels are particularly responsive to folate status. The predictive power of plasma homocysteine level as a risk factor for atherothrombotic orders raised the appealing hypothesis that reduction of homocysteine levels by vitamin supplementation might result in a commensurate reduction is the risk of atherothrombotic events. Unfortunately, most clinical trials failed to show a significant benefit of vitamin supplementation on cardiovascular events, in spite of significant lowering of plasma homocysteine levels. Thus, it is not clear whether homocysteine actually plays a causal role in many pathologies with which it is associated, or whether it is instead a marker for some other underlying mechanism. A large body of data links hyperhomocysteinemia and folate status with oxidant stress. In this article I review data that suggests that homocysteine not only promotes cellular and protein injury via oxidant mechanisms, but is also a marker for the presence of pathological oxidant stress. Thus, it is possible that hyperhomocysteinemia is not a common primary cause of atherothrombotic disorders in the general population, but rather a marker of systemic or endothelial oxidant stress that is a major mediator of these disorders.     

A cell-based model of coagulation and the role of factor VIIa

Our cell-based model of haemostasis replaces the traditional 'cascade' hypothesis, and proposes that coagulation takes place on different cell surfaces in three overlapping steps: initiation, amplification, and propagation. In highlighting the importance of cellular control during coagulation, the cell-based model allows a more thorough understanding of how haemostasis works in vivo, and sheds light on the pathophysiological mechanisms behind certain coagulation disorders. For instance, this model proposes that haemophilia involves a failure of platelet-surface FXa generation, leading to a lack of platelet-surface thrombin production. Our data suggest that high-dose FVIIa is able to bind weakly to activated platelets, independently of tissue factor, in order to generate sufficient amounts of FXa to support a burst bf thrombin generation in the absence of FIXa/FVIIIa. The considerable success of high-dose recombinant FVIIa (rFVIIa; NovoSeven, Novo Nordisk, Copenhagen, Denmark) as a therapy for patients with haemophilia and inhibitors has led to its use in a growing number of alternative indications. We believe that even in the presence of the FIXa/FVIIIa complex, rFVIIa may be able to enhance both FXa and FIXa levels on the surface of activated platelets, thus increasing the production of thrombin.     

What does it take to make the perfect clot?

The coagulation process has been conceptualized as being primarily dependent on adequate levels of the coagulation proteins. This concept was based on the clear relationship between the bleeding tendency and factor levels in hemophilia. The field is now evolving toward conceptualizing coagulation as being actively regulated by the specialized cellular components of the process. Rather than conceiving coagulation as only a "cascade" of proteolytic reactions, the coagulation reactions occur as overlapping steps on cell surfaces. Components of the old "extrinsic'" and "intrinsic" pathways of coagulation can be thought of as participating in the initiation and propagation of coagulation reactions, respectively. Thus, these pathways are not redundant as they are portrayed in the cascade model, but play distinct and complementary roles. Our understanding of how specific cellular features control the processes of hemostasis and thrombosis is developing rapidly. This review discusses some aspects of the cellular control of coagulation.     

Localization of heparin cofactor II in injured human skin: a potential role in wound healing

The physiologic function of the serpin heparin cofactor II (HCII) is not fully understood. We have hypothesized that HCII functions as an extravascular inhibitor of thrombin. Thrombin formed at a site of injury has been hypothesized to contribute to migration and proliferation of fibroblasts and smooth muscle cells involved in wound healing. To begin to test our hypothesis, we examined the immunohistochemical localization of HCII in human skin and compared it to that of the closely related serpin, antithrombin (ATIII). In skin specimens with acute wounds, there was diffuse HCII and ATIII staining in areas of hemorrhage. In healing skin wounds ATIII was primarily associated with mast cells, while HCII was associated with mononuclear phagocytes in the dermis. Blood monocytes isolated from healthy donors also stained for HCII protein. However, in situ hydridization and RT-PCR studies failed to show significant HCII mRNA expression either in macrophages in wounded skin or in peripheral blood leukocytes. HCII localization is not due to nonspecific uptake of plasma proteins, since ATIII had a very different distribution in wounded skin. These findings support the notion that HCII could function as an extravascular thrombin inhibitor and might play a role in the regulation of wound healing.     

Wound healing in hemophilia B mice and low tissue factor mice

Wound healing involves a number of physiologic mechanisms including coagulation, inflammation, formation of granulation tissue, and tissue remodeling. Coagulation with robust thrombin generation leading to fibrin formation is necessary for wound healing. It is less clear if there is a requirement for ongoing coagulation to support tissue remodeling. We have studied wound healing in mice with defects in both the initiation (low tissue factor) and propagation (hemophilia B) phases. In hemophilia B mice, dermal wound healing is delayed; this delay is associated with bleeding into the granulation tissue. Mice can be treated with replacement therapy (factor IX) or bypassing agents (factor VIIa) to restore thrombin generation. If treated just prior to wound placement, mice will have normal hemostasis in the first day of wound healing. As the therapeutic agents clear, the mice will revert to hemophilic state. If the primary role of coagulation in wound healing is to provide a stable platelet/fibrin plug that is loaded with thrombin, then treating hemophilic animals just prior to wound placement should restore normal wound healing. The results from this study did not support that hypothesis. Instead the results show that restoring thrombin generation only at the time of wound placement did not improve the delayed wound healing. In preliminary studies on low tissue factor mice, there also appears to be a delay in wound healing with evidence of bleeding into the granulation tissue. The current data suggests that ongoing coagulation function needs to be maintained to support a normal wound healing process.