Computational and Experimental Studies of the Role of Platelets in Blood Coagulation

Blood coagulation is essential for maintaining the integrity of the human vascula- ture. Whenever an injury occurs in the blood vessel, blood clots via the ‘extrinsic pathway’ (or Tissue Factor (TF) pathway) with the aid of platelets in circulation [1]. This involves the conversion of prothrombin to thrombin as the end result of cascading activation of a series of plasma serine proteases (clotting factors) that exist in a proenzyme (zymogen) form. Mechanistic models of coagulation, which incorporate the mechanisms and kinetic constants of individual reactions of the coagulation cascade, have found favor as sup- plements or even substitutes for in vitro experiments for generation of hypotheses. However, popular/recently developed mechanistic models (those in Hockin et al. [2], and Anand et al. [3], respectively) do not have equations to describe platelet activa- tion and aggregation. On the other hand, an earlier model (developed by Kuharsky & Fogelson [4]) incorporates platelets and platelet binding site densities as separate terms, but does not incorporate the latest understanding on localization of reactions (on platelet membrane or in plasma). In this thesis, the strengths of the aforemen- tioned models are combined to develop a model which is both current, and which incorporates the role of platelets and binding site densities. Platelets are an addi- tional reactant, and provide a variable concentration of reaction surfaces (in terms of platelet membranes). Coagulation reactions have been simulated at platelet concen- trations higher than as well as lower than the normal . This feature makes the model distinct from the ones mentioned earlier. Details of the model and the reaction mech- anisms used therein are discussed in Chapters 4 & 5. The model is validated, and hypothesizes that inhibition of platelet-driven activation of platelets has a more sig- nificant effect on thrombin generation than the inhibition of thrombin-driven platelet activation. A sensitivity analysis was also performed on the model to identify the reactions that the model is most and least sensitive to. The results of this study, and vii the sensitivity analysis, have been presented in Chapters 6 and 7 [5]. A model of hemostasis designed for a particular application should be reproducible, apart from making robust predictions. A modeler should heed to the importance of having consensus of kinetic constants used in the formulation of such mechanistic models. In Chapter 8, the effect of using rate constants from different experimental groups on model predictions has been studied [6] and the kinetic studies and constants for each reaction in the coagulation cascade are reviewed. This review has helped us to prepare a list of rate constants and the conditions of their applicability which can be selected based on a modeler’s requirements. Recent studies have confirmed that not all but only a small percentage of thrombin- activated platelets (“coated” platelets) exhibit procoagulant properties, that is, the expression of phosphatidylserine binding sites, required for the acceleration and progress of coagulation. In this thesis, a second mechanistic model is developed with distinct equations for phosphatidylserine-bound membrane complexes, along with a thrombin dose-dependence for the procoagulant sub-population of platelets. In addition to a significant overestimation of peak concentrations of coagulation factors Va and throm- bin, the model predicts a significantly early onset of their peak production when all activated platelets are assumed to be procoagulant instead of only a dose-dependent sub-population (that is, coated platelets). Salient features of the model and the re- sults thereof are discussed in Chapter 9. The process of fibrinolysis occurs in succession to clot formation in blood plasma. It is imperative that both the complementary processes of clot formation and lysis occur in a regulated manner in order to maintain the integrity of the human vascu- lature. Regulation of fibrinolysis in the body by constituent fibrinolysis inhibitors or by anti-fibrinoytic drugs is of importance in cases of hypocoagulation and excessive bleeding. A non-invasive method for understanding the underlying mechanisms in fibrinolysis and its inhibition by α 2-AP ( α 2 Antiplasmin), PAI-1 (Plasmin Activator viii Inhibitor-1) and TAFI (Thrombin Activable Fibrinolysis Inhibitor) is described using a mechanistic model in Chapter 10. Platelet plug formation is the first step in preventing loss of blood from the site of injury, and is followed by the appearance of a fibrin clot. The fibrin clot is the re- sultant of the coagulation cascade, and its properties are crucial to preventing blood loss: clot strength is one of the important characteristics. In this regard, a qualitative study is performed via experiments. The effect of added calcium on clot absorbance in both platelet-rich and platelet-poor plasma was investigated [7]. A 9.93% increase in the maximum absorbance is observed for platelet-rich plasma with added calcium while a 7.68% increase was observed for platelet-poor plasma. Results of this pilot study are presented in Chapter 11. The work presented in this thesis is a combination of both mathematical and ex- perimental understanding of the dynamics of blood clotting in vitro with a special emphasis on the role of platelets in the same

Effect of platelet concentration and calcium on Plasma clot absorbance

Clot structure is greatly influenced by a number of factors in vivo, calcium concentration being one of them. A pilot study with 10 healthy individuals was performed to investigate the effect of added calcium, in combination with plasma platelet concentrati on, on the turbidity of clots. Using a UV - Vis spectrophotometer, absorbance of both platelet - rich and platelet - poor clots was taken at 405 nm, in the presence as well as the absence of added calcium. A 9.93% increase in the maximum absorbance is observed for PRP with added calcium while a 7.68% increase was observed for PPP. Also, PRP clots reported higher absorbance than PPP clots both with (16.48%) and without (14.10%) calcium. It was also observed in parallel that platelet concentration also affe cted turbidity of the clots with & without added calcium

Importance of Initial Concentration of Factor VIII in a Mechanistic Model of In Vitro Coagulation

This computational study generates a hypothesis for the coagulation protein whose initial concentration greatly influences the course of coagulation. Many clinical malignancies of blood coagulation arise due to abnormal initial concentrations of coagulation factors. Sensitivity analysis of mechanistic models of blood coagulation is a convenient method to assess the effect of such abnormalities. Accordingly, the study presents sensitivity analysis, with respect to initial concentrations, of a recently developed mechanistic model of blood coagulation. Both the model and parameters to which model sensitivity is being analyzed provide newer insights into blood coagulation: the model incorporates distinct equations for plasma-phase and platelet membrane-bound species, and sensitivity to initial concentrations is a new dimension in sensitivity analysis. The results show that model predictions are most uncertain with respect to changes in initial concentration of factor VIII, and this hypothesis is supported by results from other models developed independently

Fibrinolysis: Relative Effect of Inhibitors

The process of fibrinolysis succeeds clot formation in blood plasma. It is imperative that both the complementary processes of clot formation and lysis occur in a regulated manner in order to maintain the integrity of the human vasculature. Regulation of fibrinolysis in the body by constituent fibrinolysis inhibitors or by anti-fibrinoytic drugs is of importance in cases of hypocoagulation and excessive bleeding. This study presents a non-invasive model for understanding the underlying mechanisms in fibrinolysis and its inhibition by α2AP, PAI and TAFI using a mechanistic model

Competitive Binding, and Accounting for Procoagulant Platelets, Significantly Change Thrombin Profiles in in vitro Coagulation

Background: The 'cascade' model is the accepted view for in vitro coagulation. In the cascade model, most of the coagulation reactions take place in plasma while platelets supply only amino-phospholipids for assembly of procoagulant complexes. However, the role and interaction mechanism of platelets in coagulation have been continually redefined since. Aims: Only a fraction of the activated platelets is now thought to contribute to assembly of procoagulant complexes. Further, instead of each zymogen binding to a specific number of binding sites, it is now believed that all zymogens and enzymes (except IIa) bind competitively to the shared phosphatidylserine-dependent sites on procoagulant platelets. These new findings need to be incorporated in a mechanistic model of coagulation, and their effect on thrombin profiles studied. Methods: The mechanistic model for in vitro coagulation in Susree et al (2018) incorporates a thrombin dose-dependent fraction of procoagulant platelets and competitive binding of procoagulant enzymes (except IIa) and zymogens to these platelets. Simulations of the model are compared with those from an earlier model (in Susree & Anand (2017)) with the same reactions but without these additional features. Results: Competitive binding alone (δ = 1 in Fig 1) reduces the time for peak thrombin concentration by 78.9%. Procoagulant fraction of activated platelets (δ = 0.12 in Fig 1) leads to 35.2% higher peak thrombin concentration when comparing new and old models. In the new model, peak thrombin concentration is significantly overestimated- by 299.4% and 24.7%, respectively: see Fig 2- when procoagulant fraction or thrombin dose-dependence are not included. Conclusions: Competitive binding of enzymes and zymogens, combined with thrombin dose-dependence of procoagulant platelets, causes significant qualitative and quantitative changes in the predicted thrombin profiles for in vitro coagulation. This makes a case for new hypotheses concerning role of platelets to be incorporated in mechanistic models of coagulation

Computational modeling of hypercoagulability in COVID-19

Coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has infected more than 100 million people worldwide and claimed millions of lives. While the leading cause of mortality in COVID-19 patients is the hypoxic respiratory failure from acute respiratory distress syndrome, there is accumulating evidence that shows excessive coagulation also increases the fatalities in COVID-19. Thus, there is a pressing demand to understand the association between COVID-19-induced hypercoagulability and the extent of formation of undesired blood clots. Mathematical modeling of coagulation has been used as an important tool to identify novel reaction mechanisms and to identify targets for new drugs. Here, we employ the coagulation factor data of COVID-19 patients reported from published studies as inputs for two mathematical models of coagulation to identify how the concentrations of coagulation factors change in these patients. Our simulation results show that while the levels of many of the abnormal coagulation factors measured in COVID-19 patients promote the generation of thrombin and fibrin, two key components of blood clots, the increased level of fibrinogen and then the reduced level of antithrombin are the factors most responsible for boosting the level of fibrin and thrombin, respectively. Altogether, our study demonstrates the potential of mathematical modeling to identify coagulation factors responsible for the increased clot formation in COVID-19 patients where clinical data is scarce