Factor V is activated and cleaved by platelet calpain: comparison with thrombin proteolysis

Abstract

Platelets are known to process human factor V during secretion and/or membrane binding. We studied the functional and structural changes produced in human factor V by purified human platelet calpain (calcium- activated thiol protease) and compared the alterations with those induced by thrombin. A maximum increase in coagulant activity of 2.5- fold was observed when factor V (1 U/mL, 33 nmol/L) was incubated with calpain (0.03 U/mL, 2.7 nmol/L) in comparison with a 8.8-fold increment for alpha-thrombin (0.7 U/mL, 8 nmol/L) at 25 degrees C. Thrombin additions to reactions initiated by calpain resulted in further activation comparable to that of thrombin alone, whereas the subsequent addition of calpain had no effect on the extent or pattern of the activation of factor V by thrombin. The cleavage pattern of factor V produced by these two enzymes are distinctly different. Although thrombin activation eventually results in four final components designated C1 (150 kd), D (105 kd), E (71 kd), and F1F2 (71 to 74 kd), calpain yields initial components of 200 kd and 160 kd within one minute. Further digestion of the 200 kd species by calpain gives rise first to a polypeptide of 160 kd that is converted to a 140 kd and a 120 kd species by two minutes with an increase in coagulant activity. Immunoblotting of these fragments with the monoclonal antibody (MoAb) B10 directed to factor V and the thrombin-generated C1 fragment yields results demonstrating a common epitope in these calpain-generated components of 200, 160, 140 and 120 kd. The degradation of the initial 160 kd polypeptide gives rise to polypeptides of 100 and 65 kd, both undetectable on immunoblotting with MoAb B10. The 130, 87, 58, and 48 kd components are of less certain origin. Thus, platelet calpain generates a complex but reproducible cleavage pattern different from thrombin that may explain the partial activation observed. Nevertheless, calpain processing may play a role in early hemostatic reactions involving platelets before the appearance of the first thrombin molecule.

The molecular basis of factor V and VIII procofactor activation

“…After these studies, most approaches aimed at understanding how FV is activated were principally based on correlating bond cleavage within the B‐domain with the development of procoagulant activity. These studies have largely relied on the kinetic appearance of proteolytic fragments during activation [77,79,82,83,98,99], reconstituted FV activation products [82,85], FV(a) derivatives generated by a variety of proteases [77,99–111] and recombinant FV derivatives with specific modifications to thrombin cleavage sites to establish this correlation [94,112–115]. While somewhat conflicting results have emerged, most data support the idea that variable amounts of cofactor activity will be observed depending on which region of the B‐domain is cleaved and which assay is employed to evaluate activity.…”

Thrombin-stimulated Human Platelets Express Molecular Forms of α-Granule Factor V (FV), which Differ from FVa

“…Platelet proteases may also cleave and activate FV (17)(18)(19). Bradford et al (20) documented the potential for activation of human plasma FV by calpain, a major platelet protease, and indicated that the cleavage products differed from those produced by thrombin. Platelet FV has been isolated and characterized as a partially fragmented procofactor (21).…”

Proteolysis of plasma‐derived factor V following its endocytosis by megakaryocytes forms the platelet‐derived factor V/Va pool

“…However, several other observations are inconsistent with a role for calpain in proteolysis of FV following megakaryocyte endocytosis. In contrast to megakaryocyte‐derived FV or FV/Va stored in platelets, fragments similar in size to the bonafide FVa heavy chain and light chain were not observed . Moreover, while previous observations have demonstrated that platelet calpain cleaves fibrinogen , it remains intact subsequent to its endocytosis by megakaryocytes (see Fig.…”

“…m‐ and mu‐Calpains, abundant in many cell types , are calcium‐dependent cysteine proteases that also cleave their substrates at tyrosine residues . Calpain purified from platelets cleaves plasma‐derived FV rapidly to produce several polypeptides including fragments expressing a limited amount of cofactor activity . Many of these polypeptides were similar in size to those derived from megakaryocytes.…”