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

Modepalli, Susree (2017) Computational and Experimental Studies of the Role of Platelets in Blood Coagulation. PhD thesis, Indian Insitute of Technology Hyderabad.

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Abstract

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.

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