Blood coagulation-hemostasis

Department of biochemistry
University of kerala

1. BLOOD COAGULATION By , Akash M Iyer

2. Hemostasis The term hemostasis means prevention/cessation of bleeding. Whenever a vessel is severed or ruptured, hemostasis is achieved by several mechanisms: Vascular constriction, Formation of a platelet plug, Formation of a blood clot as a result of blood coagulation, and Eventual growth of fibrous tissue into the blood clot to close the hole in the vessel permanently.

3. Blood clot

4. V ascular constriction After a blood vessel has been cut or ruptured, the trauma to the vessel wall causes the smooth muscle in the wall to contract. It is brought about by; Local myogenic spasm, Local autacoid factors from the traumatized tissues and blood platelets, Neurogenic reflexes-pain nerve impulses. The platelets are responsible for much of the vasoconstriction by releasing a vasoconstrictor substance , Thromboxane A2.

5. Primary Hemostasis

6. F ormation of Platelet Plug O verview of platelets Platelets (thrombocytes) are small anucleated cells generated from the nucleated precusor cells known as megakaryocytes in the bone marrow. Old platelets are destroyed by phagocytosis in the spleen and liver (Kupffer cells) The normal concentration ;150,000 - 300,000/μL. Components Actin and myosin molecules. Residuals of both the Endoplasmic Reticulum and Golgi apparatus. Mitochondria and enzymes that are capable of forming ATP. Fibrin-stabilizing factor. A coat of glycoproteins that repulses adherence to normal endothelium.

7. Structure of thrombocyte

8. M echanism of the Platelet Plugging When platelets come in contact with a damaged vascular surface, they immediately change their own characteristics. Begin to swell; Assume irregular forms with numerous irradiating pseudopods; Release granules that contain multiple active factors; 5- HT (Serotonin), ADP, ATP, GDP, GTP, PyroPi, histamine, fibronectin, vWF, TSP (Thrombospondin), PDGF, TGFß, VEGF, IL-ß, Factors V, XI, XIII, HMWK, fibrinogen, protein C etc. Become sticky and adhere to collagen in the tissues and to a protein called von Willebrand factor that leaks into the traumatized tissue from the plasma Secrete ADP and thromboxane A2 .

9. P latelet adhesion – a closer look As a first response to vascular injury, platelets immediately adhere to the exposed subendothelial extracellular matrix. This matrix contains several ligands for different platelet receptors, including collagen, von Willebrand factor (vWF), laminin, fibronectin and thrombospondin. Fibrillar collagens type I and III are very effective platelet activators and have a high affinity for vWF, they are considered to be the most thrombogenic matrix-mediators of platelet adhesion . Collagen Type Collagen Type I: Skin, tendon, vascular, ligature, organs, bone Collagen Type II: Cartilage Collagen Type III: Constructive fibres Collagen Type IV: Forms sources of cell basement membrane

10. vWF vWF is a large, multimeric glycoprotein that is present in plasma, the subendothelial matrix and storage granules in both platelets ( α- granules ) and endothelial cells ( Weibel-Palade bodies ). Upon injury of the vessel wall, circulating vWF rapidly binds to exposed collagen through collagen binding sites that are present in the vWF, A1 and A3 domains. After immobilization, vWF undergoes conformational changes that expose the binding site in its A1 domain for GPIbα. Platelet collagen receptor: GPVI GPVI (62 kDa) is a platelet-specific member of the IgSF. In the platelet membrane, GPVI is associated with the FcRγ -chain, which bears an ITAM for signal transduction .

11. Conformational changes in vWf allows interaction of its A3 domain with matrix collagen which induces a conformational change in the A1 domain, thereby allowing interaction with platelet receptor Gp Ib-IX-V . This interaction stimulates Ca² + release, subsequent platelet activation . Role of von Willebrand factor (vWf) in platelet adhesion

12. Platelet adhesion; Primary Hemostasis

13. When GPVI is crosslinked by collagen this leads to activation of the Src tyrosine kinases, Fyn and Lyn , bound to GPVI. The ITAMs present on the FcRγ- chain are phosphorylated by Fyn and Lyn , allowing the recruitment of the tyrosine kinase Syk. Syk in turn induces a signaling cascade finally resulting in the activation of phospholipase Cγ2 (PLCγ2). Activated PLCγ2 hydrolyzes phosphatidylinositol-4,5-bisphosphate (PIP₂, a polyphosphoinositide) to form the two internal effector molecules, Membrane bound 1,2-diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃). IP₃ rapidly diffuses and binds to its receptor IP3R, a calcium-selective channel on platelet dense tubular system, through which an efflux of Ca²+ from the DTS starts increasing Ca²+levels in the cytoplasm. Activation of Platelets Involves Stimulation of the Polyphosphoinositide Pathway

14. The hydrophobic DAG in the membrane together with Ca² + bound to phosphatidyl serine, induces the translocation of the serine/threonine protein kinase C (PKC) to the membrane. Raising cytosolic Ca²+ and DAG concentrations within the adherent platelet cytosol results in phospholipase A₂ (PLA₂) activation, platelet shape change, granule secretion and finally aggregation. Increased cytosolic Ca²+ levels also is responsible for the exposure of negatively charged phosphatidyl serine at the platelet surface due to activation of a scramblase . This negatively charged procoagulant surface provides together with bound Ca²+, binding sites for the vitamin K-dependent clotting factors, co-factors and their substrates.

15. When Ca²+ levels within the DTS are reduced following IP₃R activation, STIM1 translocates to the plasma membrane where it associates with and opens the storage operated calcium channel Orai1 .

16. Thromboxane A₂ (TxA₂) is produced as a consequence of increased Ca²+-levels that are necessary for the activation of PLA₂ by phosphorylation at  Serine-505 by P38-mitogen-activated protein kinase (MAPK). PLA₂ cleaves fatty acids from the sn-2 position in phospholipids, with e.g. the release of arachidonic acid , that itself a substrate for cyclo-oxygenase (in platelets COX-1 ). TxA ₂ induces smooth muscle cell contraction but also activates additional platelets by acting on its TP receptor .. Amplification mechanisms also operate resulting in additional platelet recruitement :

17. PLCβ2 activation, like PLCγ2, further increases cytosolic Ca²+ levels . PKC, which phosphorylates the protein pleckstrin (47 kDa); results in aggregation and release of the contents of the storage granules. IP₃ causes release of Ca² + into the cytosol mainly from the dense tubular system, which then interacts with calmodulin and myosin light chain kinase, leading to phosphorylation of the light chains of myosin. MLC phosphorylation increases actomyosin contractility and regulation of microtubule coils allowing changes in the platelet shape . ADP released from dense granules can also activate platelets, resulting in aggregation of additional platelets and provides a second feedback amplification signal by binding to the P2Y1-receptor. It also binds to P2Y12, that itself is coupled to Gαi2 (G α ), that inhibits adenylyl cyclase and hence prevents increases in cAMP generation. Gi furthermore also stimulates phosphoinositide-1,3-kinase β (PI3Kβ) that produces phosphatidyl inositol-3,4,5-trisphosphate.

18. After platelet adhesion and activation, the symmetric molecule fibrinogen cross links different platelets by binding to the activated integrin αIIbβ3 resulting in platelet aggregation. Absence of platelet aggregation due to αIIbβ3 deficiencies results in the severe bleeding disorder Glanzmann's Thrombasthenia Platelet aggregation as a result of inside-out activation

19. B lood Coagulation in the Ruptured Vessel - Formation of Blood clot It is a dynamic process of signal amplification and modulation . Activator substances from the traumatized vascular wall, from platelets, and from blood proteins adhering to the traumatized vascular wall initiate the clotting process. Once a blood clot has formed, it can follow one of two courses: It can become invaded by fibroblasts, It can dissolve Clot formation initially follows two separate pathways: Intrinsic or Contact factor pathway and Extrinsic or Tissue factor pathway These pathways merge with the formation of factor Xa, the proteinase component of the multi enzyme complex that catalyzes the formation of thrombin from prothrombin .

20. M echanism of Blood Coagulation BASIC THEORY Whether blood will coagulate depends on the balance between Procoagulants and Anticoagulants .In the blood stream, Anticoagulants normally predominate, so that the blood does not coagulate while it is circulating in the blood vessels; But when a vessel is ruptured, procoagulants from the area of tissue damage become “activated” and override the anticoagulants, and then a clot does develop. Blood Clot : The clot is composed of a meshwork of fibrin fibers running in all directions and entrapping blood cells, platelets, and plasma. The fibrin fibers also adhere to damaged surfaces of blood vessels; therefore, the blood clot becomes adherent to any vascular opening and thereby prevents further blood loss.

21. In response to rupture of the vessel or damage to the blood itself, a complex cascade of chemical reactions occurs in the blood involving more than a dozen blood coagulation factors. The net result is formation of a complex of activated substances collectively called prothrombin activator. The prothrombin activator catalyzes conversion of prothrombin into thrombin. The thrombin acts as an enzyme to convert fibrinogen into fibrin fibers that enmesh platelets, blood cells, and plasma to form a clot. Clotting process in a traumatized blood vessel

22. Initiation of Coagulation: Formation of Prothrombin Activator Trauma to the vascular wall and adjacent tissues, Trauma to the blood, or Contact of the blood with damaged endothelial cells or with collagen and other tissue elements outside the blood vessel. The intrinsic and extrinsic pathways converge in a final common pathway involving the activation of prothrombin to thrombin and the thrombin-catalyzed cleavage of fibrinogen to form the fibrin clot. In both the extrinsic and the intrinsic pathways, a series of different plasma proteins called blood clotting factors play major roles . Most of these are inactive forms of proteolytic enzymes. When converted to the active forms, their enzymatic actions cause the successive, cascading reactions of the clotting process.

23. The functions of the proteins involved in blood coagulation

24. These proteins can be classified into five types: (1) Zymogens of serine-dependent proteases , which become activated during the process of coagulation: Factor XII, Factor XI, Factor IX, Factor VII, Factor X, Factor II; (2) Cofactors ; Factor VIII, Factor V ,Tissue factor (factor III) (3) fibrinogen ; Factor I (4) a transglutaminase , which stabilizes the fibrin clot; Factor XIII and (5) Regulatory and other proteins ; Protein C, Protein S, Thrombomodulin.   Tenase (X ase ) is the final and rate-limiting enzyme complex. Extrinsic tenase complex is made up of tissue factor VII, and  Ca² +   as an activating ion. Intrinsic tenase complex contains the active factor IX (IXa), its cofactor factor VIII (VIIIa), the substrate (factor X), and they are activated by negatively charged surfaces (such as glass, active platelet membrane, These  vitamin K-dependent procoagulant factors dock to this surface through their Gla domain with Ca 2+  bridges.

25. Clot formation initially follows two separate pathways. These pathways merge with the formation of factor Xa, the proteinase component of the multienzyme complex that catalyzes the formation of thrombin from prothrombin. The clotting cascades The intrinsic cascade is initiated when contact is made between blood and exposed negatively charged surfaces. The extrinsic pathway is initiated upon vascular injury which leads to exposure of tissue factor, TF (also identified as factor III), a subendothelial cell-surface glycoprotein that binds phospholipid.  Clot Formation is a Membrane Mediated Process

26. The term intrinsic pathway because that blood clotting would occur spontaneously when blood was placed in clean glass test tubes, all components for the clotting process were intrinsic to the circulating blood. Glass contains anionic surfaces that formed the nucleation points that initiate the process. In mammals, anionic surfaces are exposed upon rupture of the endothelial lining of the blood vessels and are the binding sites for specific factors that initiate clotting in the intrinsic pathway. The Intrinsic Pathway Leads to Activation of Factor X The intrinsic pathway involves factors XII, XI, IX, VIII, and X as well as prekallikrein, HMWK, Ca²+, and platelet phospholipids. It results in the production of factor Xa (This pathway commences with the “contact phase” in which prekallikrein, HMW kininogen, factor XII, and factor XI are exposed to a negatively charged activating surface. When the components of the contact phase assemble on the activating surface, factor XII is activated to factor XIIa upon proteolysis by kallikrein. This factor XIIa, generated by kallikrein, attacks prekallikrein to generate more kallikrein, setting up a reciprocal activation. Factor XIIa, once formed, activates factor XI to XIa and also releases bradykinin from HMW kininogen. Reactions of the Intrinsic Pathway

27. . Factor XIa in the presence of Ca2+ activates factor IX (55 kDa, a zymogen containing vitamin K-dependent γ-carboxyglutamate [Gla] residues to the serine protease, factor IXa. This in turn cleaves the Arg-Ile bond in factor X (56 kDa) to produce the two chain serine protease, factor Xa . Initiation of the intrinsic pathway occurs when prekallikrein, high-molecular-weight kininogen, factor XI and factor XII are exposed to a negatively charged surface. This is termed the   contact phase   and can occur as a result of interaction with the phospholipids (primarily phosphatidyl ethanolamine , PE) of circulating lipoprotein particles such as chylomicrons, VLDLs, and oxidized LDLs. This is the basis of the role of hyperlipidemia in the promotion of a pro-thrombotic state and the development of atherosclerosis

28. Contact Activation pathway- Intrinsic Pathway

29. The activation of factor Xa requires assemblage of the  tenase complex  (Ca 2+  and factors VIIIa, IXa and X) on the surface of activated platelets. One of the responses of platelets to activation is the presentation of phosphatidylserine (PS) and phosphatidylinositol (PI) on their surfaces. The exposure of these phospholipids allows the tenase complex to form. The role of factor VIII in this process is to act as a receptor, in the form of factor VIIIa (cofactor), for factors IXa and X. The activation of factor VIII to factor VIIIa (the actual receptor) occurs in the presence of minute quantities of thrombin. As the concentration of thrombin increases, factor VIIIa is ultimately cleaved by thrombin and inactivated. This dual action of thrombin , upon factor VIII, acts to limit the extent of tenase complex formation and thus the extent of the coagulation cascade.

30. The Extrinsic Pathway Also Leads to Activation of Factor X But by a Different Mechanism The term extrinsic came from the observation that there was another factor extrinsic to circulating blood that facilitates blood clotting. This factor was identified as factor III, tissue factor The extrinsic pathway involves tissue factor, factors VII and X, and Ca²+ and results in the production of factor Xa. It is initiated at the site of tissue injury with the exposure of tissue factor on subendothelial cells. Tissue factor ( transmembrane protein) interacts with and activates factor VII (53 kDa), a circulating Gla -containing glycoprotein synthesized in the liver. Tissue factor is a cofactor in the factor VIIa-catalyzed activation of factor X. The association of tissue factor and factor VIIa is called tissue factor complex . Factor VIIa cleaves the same Arg -Ile bond in factor X that is cleaved by the tenase complex of the intrinsic pathway. A major mechanism for the inhibition of the extrinsic pathway occurs at the tissue factor-factor VIIa-Ca 2+ - Xa complex. The protein, lipoprotein-associated coagulation inhibitor,  LACI  specifically binds to this complex. LACI is also referred to as extrinsic pathway inhibitor , EPI or tissue factor pathway inhibitor, TFPI ( anticonvertin ).

31. Tissue factor pathway-Extrinsic Pathway

32. The Final Common Pathway of Blood Clotting Involves Activation of Prothrombin to Thrombin Prothrombin (72 kDa),is a single-chain glycoprotein containing ten gla residues in its N-terminal region and the serine-dependent active protease site is in the carboxyl terminal region of the molecule. Upon binding to the complex of factors Va and Xa on the platelet membrane, prothrombin is cleaved by factor Xa at two sites to generate the active, two-chain thrombin molecule, which is then released from the platelet surface. The A and B chains of thrombin are held together by a single disulfide bond. Role of Factor Va Factor V (330 kDa), a glycoprotein, synthesized in the liver, spleen, and kidney and is found in platelets as well as in plasma. It functions as a cofactor in a manner similar to that of factor VIII in the tenase complex. When activated to factor Va by traces of thrombin, it binds to specific receptors on the platelet membrane and forms a complex with factor Xa and prothrombin. It is subsequently inactivated by further action of thrombin, thereby providing a means of limiting the activation of prothrombin to thrombin.

33. Fibrinogen (factor I;340 kDa ) is a large molecule consisting of 3 pairs of polypeptides ([Aα][Bβ][γ]) 2 . The 6 chains are covalently linked near their N-terminals through disulfide bonds. The A and B portions of the Aα and Bβ chains comprise the fibrinopeptides, A and B . The fibrinopeptide regions of fibrinogen contain several glutamate and aspartate residues imparting a high negative charge to this region and aid in the solubility of fibrinogen in plasma. Active thrombin is a serine protease that hydrolyses fibrinogen at Arg-Gly (R-G) bonds between the fibrinopeptide and the A and B portions of the protein. Thrombin-mediated release of the fibrinopeptides generates fibrin monomers with a subunit structure (αβγ) 2 . These monomers spontaneously aggregate in a regular array, forming a weak fibrin clot. Activation of Fibrinogen to Fibrin

34. In addition to fibrin activation, thrombin converts factor XIII to factor XIIIa, a highly specific transglutaminase that introduces cross-links composed of covalent bonds between the amide nitrogen of glutamine and ε-amino group of lysine in the fibrin monomers. The removal of the fibrinopeptides exposes binding sites that allow the molecules of fibrin monomers to aggregate spontaneously in a regularly staggered array, yielding a more stable insoluble fibrin clot with increased resistance to proteolysis. Formation of a fibrin clot. A : Thrombin-induced cleavage of Arg-Gly bonds of the Aα and Bβ chains of fibrinogen to produce fibrinopeptides (left-hand side) and the α and β chains of fibrin monomer (right-hand side). B: Cross linking of fibrin molecules by activated factor XIII (factor XIIIa)

35. S econdary hemostasis

36. Regulation of Thrombin activity The activation of thrombin is also regulated by specific thrombin inhibitors. Antithrombin III is the most important since it can also inhibit the activities of factors IXa, Xa, XIa and XIIa, plasmin, and kallikrein. The activity of antithrombin III is potentiated in the presence of heparin by the following means: heparin binds to a specific site on antithrombin III, producing an altered conformation of the protein, and the new conformation has a higher affinity for thrombin as well as its other substrates. This effect of heparin is the basis for its clinical use as an anticoagulant. The naturally occurring heparin activator of antithrombin III is present as heparan and heparan sulfate on the surface of vessel endothelial cells.

37. Thrombin plays an important regulatory role in coagulation. Thrombin combines with thrombomodulin present on endothelial cell surfaces forming a complex that converts protein C to protein Ca . The cofactor protein S and protein C a degrade factors Va and VIIIa, thereby limiting the activity of these 2 factors in the coagulation cascade . Thrombin binds to a class of G-protein-coupled receptors (GPCRs) called protease activated receptors (PARs): PAR-1, -3 and -4. Thrombin-mediated activation of PAR-1 On the surface of platelets thrombin binds to PAR-1 resulting in release of the ligand portion of the receptor. Activation of the receptors leads to activation of G-proteins of the G q  and G 12/13  families. The response to the activated signal transduction cascades includes granule secretion, release of arachidonic acid from membrane phospholipids, and changes in cytoskeletal architecture. Role of Thrombin

38. Plasmin , a serine protease that circulates as the inactive proenzyme, plasminogen. Any free circulating plasmin is rapidly inhibited by α 2 -antiplasmin. Plasminogen binds to both fibrinogen and fibrin, thereby being incorporated into a clot as it is formed. Dissolution of Fibrin Clots Tissue plasminogen activator (tPA; alteplase) and, to a lesser degree, urokinase are serine proteases which convert plasminogen to plasmin. Inactive tPA is released from vascular endothelial cells following injury; it binds to fibrin and is consequently activated. Urokinase is produced as the precursor, prourokinase by epithelial cells lining excretory ducts. The role of urokinase is to activate the dissolution of fibrin clots that may be deposited in these ducts.

39. L evels of Circulating Thrombin Must Be Carefully Controlled or Clots May Form Once active thrombin is formed in the course of hemostasis or thrombosis, its concentration must be carefully controlled to prevent further fibrin formation or platelet activation. Four naturally occurring thrombin inhibitors exist in normal plasma. Antithrombin III , which contributes to 75% of the antithrombin activity. Antithrombin III also inhibit the activities of factors IXa, Xa, XIa, XIIa, and VIIa complexed with tissue factor. The activity of antithrombin III is greatly potentiated by the presence of acidic proteoglycans such as heparin. α -2-Macroglobulin  (inhibits fibrinolysis by inhibiting plasmin and kallikrein) contributes most of the remainder of the antithrombin activity, with heparin cofactor II and α -1-antitrypsin acting as minor inhibitors under physiologic conditions.

40. The Allosteric Role of Thrombin in Controlling Coagulation Thrombin exists in two conformational forms: one is stabilized by Na + (fast) and has high specificity for catalyzing the conversion of fibrinogen to fibrin; the other conformational form predominates in the absence of sodium (slow) , has low specificity for fibrinogen conversion, but high specificity for thrombomodulin binding and activity on protein C. Many thrombotic diseases are associated with mutations in protein C (eg; venous thromboembolism) that affect its activation by thrombin.

41. Vitamin K, (the "koagulation" vitamin) is an essential cofactor for this carboxylase Three compounds have the biologic activity of vitamin K; Phylloquinone , (phytonadione) the normal dietary source, found in green vegetables; Menaquinones , synthesized by intestinal bacteria, with differing lengths of side-chain; Menadione , Menadiol , and Menadiol diacetate , synthetic compounds that can be metabolized to phylloquinone. Vitamin K ( 2-methyl-1,4-naphthoquinones) is the cofactor for the carboxylation of glutamate residues in the post-synthetic modification of proteins to form the unusual amino acid γ-carboxyglutamate (Gla), which chelates the calcium ion. VITAMIN K VITAMERS Vitamin K vitamers and the vitamin K antagonists dicoumarol and warfarin.

42. Initially, vitamin K hydroquinone is oxidized to the epoxide which activates a glutamate residue in the protein substrate to a carbanion, that reacts non-enzymically with CO₂ to form γ-carboxyglutamate . Vitamin K epoxide is reduced to the quinone form by  vitamin K epoxide reductase (VKORC1) , and the quinone is reduced to the active hydroquinone form by either the same VKORC1 or vitamin K quinone reductase (VKQR ). These latter two reactions involve a dithiol conversion to a disulfide. The role of vitamin K in the biosynthesis of γ- carboxyglutamate .

43. As a biologically active barrier, the endothelium is semi-permeable and regulates the transfer of small and large molecules. Endothelial cells have a role in maintaining a non-thrombogenic blood tissue interface and regulate thrombosis, thrombolysis, platelet adherence, vascular tone and blood flow. Key processes to prevent platelet activation (and therefore coagulation) include inactivation of thrombin, conversion of ATP to inert AMP through the action of ATPases and ADPases, and blocking the physical interaction between platelets and collagen, which can activate platelets. E ndothelial Cells Line All Blood Vessels

44. Anticoagulant activities of endothelium Endothelial cells bind and display tissue factor pathway inhibitors (TFPIs). Endothelial cells synthesize and display heparan sulphate proteoglycans (HS) on their cell surface. Endothelial cells also synthesize and display the protein thrombomodulin, which binds thrombin and converts its substrate specificity from cleavage of fibrinogen to cleavage and activation of protein C. Activated protein C is an enzyme that destroys certain clotting factors and inhibits coagulation . Endothelial cells also sequester von Willebrand factor (vWF), a protein that strengthens the interaction of platelets with the basement membrane, by keeping it within their storage granules, known as Weibel–Palade bodies (WPB). Nitric oxide (NO), generated by nitric-oxide synthase 3 (NOS3)-mediated conversion of arginine, further inhibits platelet activation

45. A thrombus is a blood clot anchored to damaged vascular wall. The thrombus formation process in arteries can lead to heart attacks or ischemic strokes if the affected arteries are the coronary or the carotids. Intracavitary thrombus can also be dislodged from the heart and be embolized to the brain producing cardiogenic strokes. In  thromboembolism , the thrombus (blood clot) from a blood vessel is completely or partially detached from the site of thrombosis (clot). The blood flow will then carry the embolus (via blood vessels) to various parts of the body where it can block the lumen (vessel cavity) and cause vessel obstruction or occlusion. Thrombosis

46. Virchow's triad of hypercoagulability, venous stasis, and injury to the vessel wall provides a model for understanding many of the risk factors that lead to the formation of thrombosis.

47. REFERENCES Harper’s Illustrated Biochemistry . 29th ed. New York: McGraw-Hill. Integrative Medical Biochemistry : Examination and Board Review Michael W. King McGraw Hill Professional. Textbook of Biochemistry with Clinical Correlations : Fourth Edition Edited by Thomas M. Devlin WileyLiss, Inc. Textbook of medical physiology - Arthur C. Guyton, John E. Hall.—11th ed. Elsevier Inc. Blood platelet biochemistry Article in Thrombosis Research - November 2011. Platelets: Still a Therapeutical Target for Haemostatic Disorders International Journal of Molecular Sciences 2014. Platelet shape change and spreading - Methods in Molecular Biology – Springer 2012. A Review of Macroscopic Thrombus Modeling Article in Thrombosis Research · December 2012.

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