Factor‐V expression in platelets from human megakaryocytic culture

Abstract

Summary The origin of platelet‐factor‐V has long been discussed. To elucidate whether and when human platelet‐factor‐V is synthesized by megakaryocytes, we utilized in vitro‐generated megakaryocytes capable of producing platelets. Factor‐V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor‐V protein was expressed in mature proplatelet‐bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor‐V.

Residual platelet factor V ensures thrombin generation in patients with severe congenital factor V deficiency and mild bleeding symptoms

“…3,4 FVa activity is down-regulated by activated protein C (APC), which inactivates FVa by cleaving the heavy chain at Arg 306 , Arg 506 , and Arg 679 . 5,6 Although megakaryocytes are capable of FV synthesis, 7,8 platelet FV originates from the plasma pool. [9][10][11] Bone marrow megakaryocytes internalize plasma FV via a specific receptormediated process 12 and store it in secretory ␣-granules.…”

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation

“…Although megakaryocytes are not the physiological site of FV production in vivo, [5][6][7] they have been shown to synthesize FV in culture, 4 offering us the opportunity to test the antisense molecules on the patient's own cells without a liver biopsy. Unfortunately, the antisense molecules could not be tested directly on the patient's hepatocytes, but their efficacy in correcting the splicing defect in HepG2 cells transfected with the mutant F5 minigene construct suggests that they would be able to restore FV expression in the patient's hepatocytes as well, especially considering the fact that the liver is a preferential site of accumulation of systemically injected MOs.…”

“…3 Although megakaryocytes can synthesize FV,4 platelet FV originates from secondary endocytosis of plasma FV. [5][6][7] FV deficiency 8-10 is a rare bleeding disorder inherited as an autosomal-recessive trait and is associated with mutations in the F5 gene.…”

A novel in-frame deletion in the factor V C1 domain associated with severe coagulation factor V deficiency in a Korean family

“…Factor V is composed of six domains named sequentially A1, A2, B, A3, C1, and C2 from the N-terminal to the C-terminal end [1,2]. Synthesized mainly by hepatocytes, about 20% of factor V is stored into platelets a-granules [3], whereas the remaining 80% circulates in the bloodstream as an inactive cofactor. Factor V activation is mediated by thrombin or FXa with cleavage at three arginine residues (Arg709, Arg1018, and Arg1545) and subsequent removal of the protruding B domain.…”