Abstract
Protein Z (PZ) is a vitamin K-dependent protein, whereas PZ-dependent protease inhibitor (ZPI) is a member of the serine protease inhibitor superfamily. ZPI rapidly inhibits factor Xa in the presence of PZ, calcium, and phospholipids and inhibits factor XIa in a PZ-independent fashion (Blood2000; 96:3049–3055). PZ circulates as a complex with ZPI in plasma and deletion of either the PZ or the ZPI gene is associated with the prothrombotic phenotype in mice. In addition, W303X or R67X nonsense mutations in the ZPI gene are reportedly associated with deep venous thrombosis in certain human populations. Western blot analysis of platelets stimulated with thrombin (0–200 mU/mL) showed they contained and released ZPI (approximately 200 ng/109 platelets) with the same molecular weight as plasma ZPI (72 kDa). The majority of the ZPI was released within 1 min. by 25 mU/mL thrombin. PZ was not detected in platelets by western blot analysis. Immunohistochemical staining using a monoclonal anti-ZPI antibody demonstrated a cytoplasmic fine granular staining pattern in maturing megakaryocytes in bone marrow aspirates and in circulating platelets, suggesting that ZPI may be stored in alpha granules. ZPI mRNA, however, was not detected by reverse transcriptase polymerase chain reaction (RT-PCR) in platelets or bone marrow aspirates, but was detected in human liver cDNA. RT-PCR for platelet factor 4 and glyceraldehyde 3-phosphate dehydrogenase mRNA showed amplified products with expected sizes. In conclusion, thrombin-releasable ZPI, but not PZ, is present in platelets and is most likely derived from the uptake of ZPI from plasma. ZPI released from activated platelets may play a role in the regulation of local coagulation at a site of injury.
Engineering a protein Z‐dependent protease inhibitor (
ZPI
) mutant as a novel antagonist of
ZPI
anticoagulant function for hemophilia treatment
