Activated platelets bind numerous adhesive and procoagulant proteins by receptor-mediated processes. Although there is little evidence to suggest that these processes are heterogeneous in platelets, we previously found that platelets co-stimulated with collagen and thrombin express functional alpha-granule factor V only on a subpopulation of cells. Here we show that these cells, referred to as 'COAT-platelets', bind additional alpha-granule proteins, including fibrinogen, von Willebrand factor, thrombospondin, fibronectin and alpha2-antiplasmin. These proteins are all transglutaminase substrates, and inhibitors of transglutaminase prevent the production of COAT-platelets. A synthetic transglutaminase substrate (CP15) also binds to COAT-platelets, and analysis by high performance liquid chromatography/mass spectrometry shows that a product is formed with a relative molecular mass (Mr) equal to CP15 plus 176. Serotonin, an abundant component of platelet-dense granules, has an Mr of 176, and fibrinogen isolated from COAT-platelets contains covalently linked serotonin. Synthetic bovine serum albumin-(serotonin)6 binds selectively to COAT-platelets and also inhibits the retention of procoagulant proteins on COAT-platelets. These data indicate that COAT-platelets use serotonin conjugation to augment the retention of procoagulant proteins on their cell surface through an as yet unidentified serotonin receptor.

The human ELL gene on chromosome 19 undergoes frequent translocations with the trithorax-like MLL gene on chromosome 11 in acute myeloid leukemias. Here, ELL was shown to encode a previously uncharacterized elongation factor that can increase the catalytic rate of RNA polymerase II transcription by suppressing transient pausing by polymerase at multiple sites along the DNA. Functionally, ELL resembles Elongin (SIII), a transcription elongation factor regulated by the product of the von Hippel-Lindau (VHL) tumor suppressor gene. The discovery of a second elongation factor implicated in oncogenesis provides further support for a close connection between the regulation of transcription elongation and cell growth.

Human ␣ 2 -antiplasmin (␣ 2 AP), also known as ␣ 2 -plasmin inhibitor, is the major inhibitor of the proteolytic enzyme plasmin that digests fibrin. There are 2 N-terminal forms of ␣ 2 AP that circulate in human plasma: a 464-residue protein with Met as the N-terminus, Met-␣ 2 AP, and a 452-residue version with Asn as the N-terminus, Asn-␣ 2 AP. We have discovered and purified a proteinase from human plasma that cleaves the Pro12-Asn13 bond of Met-␣ 2 AP to yield Asn-␣ 2 AP and have named it antiplasmin-cleaving enzyme (APCE). APCE is similar in primary structure and catalytic properties to membranebound fibroblast activation protein/seprase for which a physiologic substrate has not been clearly defined. We found that Asn-␣ 2 AP becomes cross-linked to fibrin by activated factor XIII approximately 13 times faster than native Met-␣ 2 AP during clot formation and that clot lysis rates are slowed in direct proportion to the ratio of Asn-␣ 2 AP to Met-␣ 2 AP in human plasma. We conclude that APCE cleaves Met-␣ 2 AP to the derivative Asn-␣ 2 AP, which is more efficiently incorporated into fibrin and consequently makes it strikingly resistant to plasmin digestion. APCE may represent a new target for pharmacologic inhibition, since less generation and incorporation of Asn-␣ 2 AP could result in a more rapid removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states. IntroductionHuman ␣ 2 -antiplasmin (␣ 2 AP), also known as ␣ 2 -plasmin inhibitor, is the main inhibitor of plasmin. 1 Plasmin plays a critical role in fibrin proteolysis and tissue remodeling. The physiologic relevance of plasmin inhibition by ␣ 2 AP to blood clotting and fibrinolytic homeostasis is supported by the following observations: (1) the rate of free plasmin inactivation by circulating ␣ 2 AP is much faster than fibrin(ogen) digestion by plasmin, 2 thereby eliminating the possibility of a systemic lytic state and consequent bleeding; (2) ␣ 2 AP is cross-linked to forming fibrin by activated blood clotting factor XIII (FXIIIa) and inhibits plasmin-mediated lysis in direct proportion to the amount incorporated 3-5 ; and (3) patients with homozygous ␣ 2 AP deficiency manifest serious hemorrhagic tendencies, while heterozygotes tend to bleed only after major trauma or surgery. 6 Human ␣ 2 AP is synthesized primarily in the liver, and during circulation in plasma, the secreted precursive Met-␣ 2 AP form, a 464-residue protein having Met as the N-terminus, undergoes proteolytic cleavage between Pro12 and Asn13 to yield Asn-␣ 2 AP, a 452-residue version with Asn as the N-terminus. 7 Met-␣ 2 AP accounts for approximately 30% of circulating ␣ 2 AP, and Asn-␣ 2 AP, approximately 70%. 7,8 While 3-fold more Asn-␣ 2 AP than recombinant Met-␣ 2 AP was shown to cross-link to fibrin, 9 no data have been reported for native circulating Met-␣ 2 AP. Moreover, the effect of different ratios of the 2 ␣ 2 AP forms on clot lysis has not been reported. Finally, the enzyme responsible for converting Met-␣ 2 AP to Asn-␣ 2 AP has not been ident...

Circulating antiplasmin-cleaving enzyme (APCE) has a role in fibrinolysis and appears structurally similar to fibroblast activation protein (FAP), a cell-surface proteinase that promotes invasiveness of certain epithelial cancers. To explore this potential relationship, we performed comparative structure/function analyses of the 2 enzymes. APCE from human plasma and recombinant FAP (rFAP) exhibited identical pH optima of 7.5, extinction coefficients (

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