Platelets express steroidogenic 17beta-hydroxysteroid dehydrogenases. Distinct profiles predict the essential thrombocythemic phenotype.

Human blood platelets have important, regulatory functions in diverse hemostatic and pathological disorders, including vascular remodeling, inflammation, and wound repair. Microarray analysis was used to study the molecular basis of essential thrombocythemia, a myeloproliferative disorder with quantitative and qualitative platelet defects associated with cardiovascular and thrombohemorrhagic symptoms, not infrequently neurological. A platelet-expressed gene (HSD17B3) encoding type 3 17beta-hydroxysteroid dehydrogenase (previously characterized as a testis-specific enzyme catalyzing the final step in gonadal synthesis of testosterone) was selectively down-regulated in ET platelets, with reciprocal induction of the type 12 enzyme (HSD17B12). Functional 17beta-HSD3 activity corresponding to approximately 10% of that found in murine testis was demonstrated in normal platelets. The induction of HSD17B12 in ET platelets was unassociated with a concomitant increase in androgen biosynthesis, suggesting distinct functions and/or substrate specificities of the types 3 and 12 enzymes. Application of a molecular assay distinguished ET from normal platelets in 20 consecutive patients (p < 0.0001). These data provide the first evidence that distinct subtypes of steroidogenic 17beta-HSDs are functionally present in human blood platelets, and that the expression patterns of HSD17B3 and HSD17B12 are associated with an uncommon platelet disorder manifest by quantitative and qualitative platelet defects.

Expression of protease activated receptor 3 (PAR3) is upregulated by induction of megakaryocyte phenotype in human erythroleukemia (HEL) cells.

OBJECTIVE: Two major protease-activated receptors (PARs), PAR1 and PAR4, are involved in the activation of human platelets by thrombin. A third, PAR3, is preferentially expressed by tissues of hematopoietic origin and megakaryocytes. Although PAR3 is also a thrombin substrate, its low-level expression on human platelets suggests a function distinct from that of PAR1, the major receptor involved in thrombin-mediated platelet activation. We studied the expression of PARs during megakaryocyte differentiation of human erythroleukemia (HEL) cells in order to determine the role of PAR3 in megakaryocytopoiesis. METHODS: HEL cells exposed to phorbol 12-myristate 13-acetate (PMA) to induce megakaryocyte differentiation were examined by light microscopy and flow cytometry (DNA ploidy, surface expression of PAR1, PAR3, GPIIb-IIIa). Northern blot, RT-PCR, and quantitative RT-PCR were used to evaluate the expression of PARs 1, 3, and 4 mRNA. HEL cells were also exposed to thrombin and thrombopoietin (TPO). RESULTS: In baseline studies, unstimulated HEL cells were found to express comparable levels of PAR1 and PAR3 by Northern blot. Minimal expression of PAR4 was detected by RT-PCR, but not by Northern analysis. Exposure to PMA, but not thrombin or TPO, resulted in megakaryocytic differentiation as evident by increased cell size and nuclear complexity, increased ploidy, and enhanced expression of GPIIb-IIIa, a specific marker of megakaryocytes/platelets. PMA-stimulated HEL cells showed enhanced PAR3 cell-surface expression (approximately threefold increase by day 2) by flow cytometry. In contrast, there was no change in cell-surface PAR1 expression. Northern blot analysis (approximately 10-fold) and quantitative RT-PCR (approximately threefold) confirmed the upregulation of PAR3 mRNA expression (by 24 hours) in cells exposed to PMA. This did not occur with exposure to TPO. CONCLUSION: These data demonstrate increased expression of PAR3 mRNA and protein in HEL cells undergoing megakaryocytic maturation following PMA exposure, suggesting a developmental role for PAR3. Furthermore, regulation of PAR3 expression appears to be specifically coupled to the protein kinase C system, but independent of the Ras/Raf/MAP kinase pathway.

Distinct PAR/IQGAP expression patterns during murine development: implications for thrombin-associated cytoskeletal reorganization.

Thrombin has a critical role in many adult and embryologic cellular processes, exerting its effects through two high-affinity thrombin receptor systems: protease-activated receptor 1 (PAR1) and the PAR3/PAR4 system. Both hPAR1 and hPAR3 are coclustered in the human genome, with hPAR3 encompassed within hIQGAP2, a putative GTPase activating protein with actin polymerizing functions linked to cytoskeletal reorganization. Since hPARs colocalize with hIQGAP2 in the human genome and function coordinately with this protein in platelet thrombin signaling pathways, we have further characterized these genes in developing embryonic and adult tissues. We confirmed the presence of a mIQGAP2/ mPAR gene cluster on murine Chromosome 13 and showed it to be organized similarly to that in humans, except that murine PAR3 is translated off the forward (sense) strand. Northern analysis demonstrated limited mPAR3 expression in adult tissues, although its expression during embryogenesis was evident at E15 in cartilage, brain, and keratinocytes. mIQGAPs 1 and 2 had congruent expression patterns in 11 of 15 adult tissues studied. In contrast, whole embryos demonstrated predominant mIQGAP1 expression starting at E7 and evident to E17. In situ hybridization of whole embryos (E9-E16) demonstrated distinct patterns of tissue-dependent mIQGAP1/ mIQGAP2 expression. Concordant expression (absence or presence) of mPAR1 with either mIQGAP1 or mIQGAP2 was seen in the majority (12 of 15) of adult tissues studied. Similarly, there was no evidence for mPAR3 expression during embryogenesis in the absence of either mIQGAP1 or mIQGAP2. These data provide a panoramic survey of PAR/ IQGAP expression as an initial approach to dissect thrombin signaling pathways linked to cytoskeletal reorganization.

IQGAP2 functions as a GTP-dependent effector protein in thrombin-induced platelet cytoskeletal reorganization.

Human blood platelets are anucleate cells whose response to extracellular stimuli results in actin cytoskeleton rearrangements, thereby providing the critical initial step in the regulation of hemostasis. The serine protease alpha-thrombin, known to activate platelets by cleavage of a family of protease-activated receptors (PARs), is the most potent physiologic activator of human platelets, though downstream effector proteins uniquely linked to platelet cytoskeletal actin polymerization remain largely uncharacterized. The gene encoding the putative rac1/cdc42 effector protein IQGAP2 was identified within the PAR gene cluster at 5q13, flanked telomeric by PAR1 and encompassing PAR3. Immunofluorescence microscopy demonstrated IQGAP2 expression in filopodial extensions of activated platelets and colocalized with F-actin in lamellipodia and filopodia of IQGAP2-transfected COS1 cells. Platelet activation by alpha-thrombin, but not saturating concentrations of fibrillar collagen or adenosine 5'-diphosphate, uniquely assemble an IQGAP2/arp2/3-actin cytoplasmic complex, an association regulated by guanosine triphosphate rac1 ([GTP]rac1) but not by [GTP]cdc42. Likewise, only thrombin-activated platelets resulted in rapid translocation of IQGAP2 to the platelet cytoskeleton. These observations identify a physiologic scaffolding function for IQGAP2 and establish the presence of a functional genomic unit in humans uniquely evolved to regulate thrombin-induced platelet cytoskeletal actin reorganization.