Rap1b In Platelets Research Articles

Vasodilator-Stimulated Phosphoprotein (VASP)-dependent and -independent pathways regulate thrombin-induced activation of Rap1b in platelets.

BackgroundVasodilator-Stimulated Phosphoprotein (VASP) is involved in the inhibition of agonist-induced platelet aggregation by cyclic nucleotides and the adhesion of platelets to the vascular wall. αIIbβ3 is the main integrin responsible for platelet activation and Rap1b plays a key role in integrin signalling. We investigated whether VASP is involved in the regulation of Rap1b in platelets since VASP-null platelets exhibit augmented adhesion to endothelial cells in vivo.MethodsWashed platelets from wild type and VASP-deficient mice were stimulated with thrombin, the purinergic receptors agonist ADP, or the thromboxane A2 receptor agonist U46619 and Rap1b activation was measured using the GST-RalGDS-RBD binding assay. Interaction of VASP and Crkl was investigated by co-immunoprecipitation, confocal microscopy, and pull-down assays using Crkl domains expressed as GST-fusion proteins.ResultsSurprisingly, we found that activation of Rap1b in response to thrombin, ADP, or U46619 was significantly reduced in platelets from VASP-null mice compared to platelets from wild type mice. However, inhibition of thrombin-induced activation of Rap1b by nitric oxide (NO) was similar in platelets from wild type and VASP-null mice indicating that the NO/cGMP/PKG pathway controls inhibition of Rap1b independently from VASP. To understand how VASP regulated Rap1b, we investigated association between VASP and the Crk-like protein (Crkl), an adapter protein which activates the Rap1b guanine nucleotide exchange factor C3G. We demonstrated the formation of a Crkl/VASP complex by showing that: 1) Crkl co-immunoprecipitated VASP from platelet lysates; 2) Crkl and VASP dynamically co-localized at actin-rich protrusions reminiscent of focal adhesions, filopodia, and lamellipodia upon platelet spreading on fibronectin; 3) recombinant VASP bound directly to the N-terminal SH3 domain of Crkl; 4) Protein Kinase A (PKA) -mediated VASP phosphorylation on Ser157 abrogated the binding of Crkl.ConclusionsWe identified Crkl as a novel protein interacting with VASP in platelets. We propose that the C3G/Crkl/VASP complex plays a role in the regulation of Rap1b and this explains, at least in part, the reduced agonist-induced activation of Rap1b in VASP-null platelets. In addition, the fact that PKA-dependent VASP phosphorylation abrogated its interaction with Crkl may provide, at least in part, a rationale for the PKA-dependent inhibition of Rap1b and platelet aggregation.

Differential regulation of platelet inhibition by cGMP- and cAMP-dependent protein kinases

Background Under normal conditions circulating platelets are maintained in a resting state by the paracrine signaling molecules, prostacyclin (PGI2) and nitric oxide (NO), released from vascular endothelial cells. PGI2 and NO trigger the synthesis of cAMP and cGMP, respectively, which in turn leads to the activation of Protein Kinase A (PKA) and Protein Kinase G (PKG). These cyclic nucleotide-dependent protein kinases phosphorylate numerous substrate proteins involved in several aspects of platelet activation. Both, PKA and PKG can phosphorylate the same substrates including VASP, LASP, RAP1GAP2, Rap1B, HSP27, and some others. Only few substrates are known to be specific for PKA (PDE3) and PKG (PDE5) [1,2]. Here we report the identification and characterization of a new PKA specific substrate, CalDAG-GEFI (calcium and diacylglycerol regulated guanine nucleotide exchange factor I), a guanine exchange factor for Rap1b in platelets.

Phosphorylation of CalDAG‐GEFI by protein kinase A prevents Rap1b activation

Signaling via protein kinase A (PKA) and protein kinase G (PKG) is critical for maintaining platelets in the resting state. Both kinases down-regulate the activity of the small GTPase Rap1b, a critical signaling switch for integrin activation and platelet aggregation. However, the mechanism of Rap1b regulation by PKA and PKG is largely unknown. To identify the PKA phosphorylation sites in calcium and diacylglycerol-regulated guanine nucleotide exchange factor I (CalDAG-GEFI), the main GEF for Rap1b in platelets, and the effect of CalDAG-GEFI phosphorylation in Rap1b activation. The phosphorylation sites in CalDAG-GEFI were identified by radio-active phosphate incorporation assay and mass spectrometry. Phospho-antibody was developed to detect CalDAG-GEFI phosphorylation in Western blots. Rap1b activation was detected by Rap1-GTP pull-down assay. S587 was identified as the major PKA phosphorylation site in CalDAG-GEFI, while S116/117 was weakly phosphorylated. Phosphorylation of S587 correlated with the inhibitory effect of PKA on Rap1b activation in platelets. In HEK293 cells, expression of a phospho-mimetic mutant of CalDAG-GEFI (S587D) abolished agonist-induced Rap1b activation. Mutation of S587 to alanine partially reversed the inhibitory effect of PKA signaling on Rap1b activation, while mutation of S116, S117 and S587 to alanine completely abolished the inhibitory effect of PKA on Rap1b activation. Our study strongly suggests that phosphorylation of CalDAG-GEFI is a critical mechanism by which PKA controls Rap1b-dependent platelet aggregation.

Phosphorylation of the guanine-nucleotide-exchange factor CalDAG-GEFI by protein kinase A regulates Ca2+-dependent activation of platelet Rap1b GTPase

In blood platelets the small GTPase Rap1b is activated by cytosolic Ca2+ and promotes integrin αIIbβ3 inside-out activation and platelet aggregation. cAMP is the major inhibitor of platelet function and antagonizes Rap1b stimulation through a mechanism that remains unclear. In the present study we demonstrate that the Ca2+-dependent exchange factor for Rap1b, CalDAG-GEFI (calcium and diacylglycerol-regulated guanine-nucleotide-exchange factor I), is a novel substrate for the cAMP-activated PKA (protein kinase A). CalDAG-GEFI phosphorylation occurred in intact platelets treated with the cAMP-increasing agent forskolin and was inhibited by the PKA inhibitor H89. Purified recombinant CalDAG-GEFI was also phosphorylated invitro by the PKA catalytic subunit. By screening a panel of specific serine to alanine residue mutants, we identified Ser116 and Ser586 as PKA phosphorylation sites in CalDAG-GEFI. In transfected HEK (human embryonic kidney)-293 cells, as well as in platelets, forskolin-induced phosphorylation of CalDAG-GEFI prevented the activation of Rap1b induced by the Ca2+ ionophore A23187. In platelets this effect was associated with the inhibition of aggregation. Moreover, cAMP-mediated inhibition of Rap1b was lost in HEK-293 cells transfected with a double mutant of CalDAG-GEFI unable to be phosphorylated by PKA. The results of the present study demonstrate that phosphorylation of CalDAG-GEFI by PKA affects its activity and represents a novel mechanism for cAMP-mediated inhibition of Rap1b in platelets.

DISTINCT ROLES for Rap1b In PLATELET SECRETION and INTEGRIN aIIBb3 OUTSIDE-In SIGNALING

Abstract 2200Rap1b is activated by platelet agonists, and plays a critical role in integrin aIIbb3 inside-out signaling and platelet aggregation. In this study, we identify two novel functions of Rap1b in platelets. We show that agonist-induced Rap1b activation plays an important role in stimulating secretion of platelet granules. We also show that aIIbb3 outside-in signaling can activate Rap1b, and integrin outside-in signaling-mediated Rap1b activation is important in facilitating platelet spreading on fibrinogen and clot retraction. Rap1b deficient platelets had diminished ATP secretion and P-selectin expression induced by thrombin or collagen. Defect in secretion of Rap1b deficient platelets was not due to reduced granule contents, because the amount of serotonin (5HT) and platelet factor 4 (PF4) in Rap1b deficient platelets is similar to that in wild type platelets. Data from transmission electron microscopy indicate that under resting conditions, wild type and Rap1b deficient platelets had normal discoid shapes with similar numbers of granules. When stimulated with thrombin, wild type platelets showed an irregular appearance with protruding filopodia and lack of granules. In contrast, thrombin-stimulated Rap1b−/– platelets showed that more Rap1b−/– platelets contained visible a granules than wild type platelets. Dense granules were also more obvious in thrombin-stimulated Rap1b−/– platelets than wild type platelets. Importantly, addition of low doses of ADP and/or fibrinogen restored aggregation of Rap1b deficient platelets. Furthermore, we found that Rap1b was activated by platelet spreading on immobilized fibrinogen, a process that was not affected by P2Y12 or TP deficiency, but was inhibited by the selective Src inhibitor PP2, the PKC inhibitor Ro-31-8220, or the calcium chelator demethyl-BAPTA. Clot retraction was abolished, and platelet spreading on fibrinogen was diminished in Rap1b deficient platelets compared with wild type controls. The defects in clot retraction and spreading on fibrinogen of Rap1b deficient platelets were not rescued by addition of MnCl2, which elicits aIIbb3 outside-in signaling in the absence of inside-out signaling. Thus, our results reveal two different activation mechanisms of Rap1b as well as novel functions of Rap1b in platelet secretion and in integrin aIIbb3 outside-in signaling. Disclosures:No relevant conflicts of interest to declare.

Thrombopoietin Complements Gi- but Not Gq-dependent Pathways for Integrin αIIbβ3 Activation and Platelet Aggregation

Binding of thrombopoietin (TPO) to the cMpl receptor on human platelets potentiates aggregation induced by a number of agonists, including ADP. In this work, we found that TPO was able to restore ADP-induced platelet aggregation upon blockade of the G(q)-coupled P2Y1 purinergic receptor but not upon inhibition of the G(i)-coupled P2Y12 receptor. Moreover, TPO triggered platelet aggregation upon co-stimulation of G(z) by epinephrine but not upon co-stimulation of G(q) by the thromboxane analogue U46619. Platelet aggregation induced by TPO and G(i) stimulation was biphasic, and cyclooxygenase inhibitors prevented the second but not the first phase. In contrast to ADP, TPO was unable to induce integrin alpha(IIb)beta(3) activation, as evaluated by binding of both fibrinogen and PAC-1 monoclonal antibody. However, ADP-induced activation of integrin alpha(IIb)beta(3) was blocked by antagonists of the G(q)-coupled P2Y1 receptor but was completely restored by the simultaneous co-stimulation of cMpl receptor by TPO. Inside-out activation of integrin alpha(IIb)beta(3) induced by TPO and G(i) stimulation occurred independently of thromboxane A(2) production and was not mediated by protein kinase C, MAP kinases, or Rho-dependent kinase. Importantly, TPO and G(i) activation of integrin alpha(IIb)beta(3) was suppressed by wortmannin and Ly294002, suggesting a critical regulation by phosphatidylinositol 3-kinase. We found that TPO did not activate phospholipase C in human platelets and was unable to restore ADP-induced phospholipase C activation upon blockade of the G(q)-coupled P2Y1 receptor. TPO induced a rapid and sustained activation of the small GTPase Rap1B through a pathway dependent on phosphatidylinositol 3-kinase. In ADP-stimulated platelets, Rap1B activation was reduced, although not abolished, upon blockade of the P2Y1 receptor. However, accumulation of GTP-bound Rap1B in platelets activated by co-stimulation of cMpl and P2Y12 receptor was identical to that induced by the simultaneous ligation of P2Y1 and P2Y12 receptor by ADP. These results indicate that TPO can integrate G(i), but not G(q), stimulation and can efficiently support integrin alpha(IIb)beta(3) activation platelet aggregation by an alternative signaling pathway independent of phospholipase C but involving the phosphatidylinositol 3-kinase and the small GTPase Rap1B.

Mechanisms for Thrombopoietin-Induced Potentiation of Platelet Aggregation.

Thrombopoietin (TPO) is the major cytokine regulating megakaryocytes proliferation and thrombopoiesis. Binding of TPO to the c-Mpl receptor expressed on human platelets promotes tyrosine phosphorylation of a number of proteins and potentiates platelet aggregation induced by low concentrations of a variety of agonists including ADP. Nevertheless, TPO alone is not able to induce any platelet functional response, such as shape change, granule secretion, or aggregation. It is now clear that platelet aggregation induced by many different agonists results from the concomitant activation of both Gi-and Gq-dependent signaling pathways. In particular, activation of a Gi-dependent pathway is essential, albeit not sufficient, to elicit complete platelet aggregation. Recently, it has been proposed that the small GTPase Rap1B is an important element in the Gi-dependent pathway for platelet aggregation. ADP activates Gq and Gi-dependent pathways by binding to the P2Y1 and the P2Y12 receptors, respectively. In this study we investigated the contribution of TPO in platelet activation induced by ADP. We found that TPO could restore ADP-induced platelet aggregation when the Gq-coupled receptor was blocked, but failed to restore ADP-induced aggregation in absence of Gi stimulation. Similarly, TPO was unable to restore U46619-induced platelet aggregation, when the Gi-coupled P2Y12 receptor for ADP was blocked, but caused irreversible platelet aggregation when added in combination with epinephrine, which binds to a Gi-coupled receptor. TPO did not cause any detectable calcium movements or pleckstrin phosphorylation in human platelets. Moreover, TPO failed to restore ADP-induced cytosolic calcium incresases and pleckstrin phosphorylation when the Gq-coupled P2Y1 receptor was blocked, although aggregation was completely restored. Moreover, we found that TPO induced activation of Rap1B in platelets, and could completely restore ADP-induced Rap1B activation, which was partially reduced upon blockade of the P2Y1 receptor. Finally, ADP-induced binding of both fibrinogen and PAC-1 monoclonal antibody to integrin αIIbβ3 was prevented by the blockade of the Gq-coupled P2Y1 receptor, but completely restored by the simultaneous addition of TPO. These findings indicate that TPO can complement Gi-, but not Gq-dependent pathways for integrin αIIbβ3 activation and platelet aggregation, through a new mechanism that does not involve activation of phospholipase C, but may involve the small GTPase Rap1B.

Contribution of Protease-activated Receptors 1 and 4 and Glycoprotein Ib-IX-V in the Gi-independent Activation of Platelet Rap1B by Thrombin

Thrombin activates human platelets through three different membrane receptors, the protease-activated receptors PAR-1 and PAR-4 and the glycoprotein Ib (GPIb)-IX-V complex. We investigated the contribution of these three receptors to thrombin-induced activation of the small GTPase Rap1B. We found that, similarly to thrombin, selective stimulation of either PAR-1 or PAR-4 by specific activating peptides caused accumulation of GTP-bound Rap1B in a dose-dependent manner. By contrast, in PAR-1- and PAR-4-desensitized platelets, thrombin failed to activate Rap1B. Thrombin, PAR-1-, or PAR-4-activating peptides also induced the increase of intracellular Ca(2+) concentration and the release of serotonin in a dose-dependent manner. We found that activation of Rap1B by selected doses of agonists able to elicit comparable intracellular Ca(2+) increase and serotonin release was differently dependent on secreted ADP. In the presence of the ADP scavengers apyrase or phosphocreatine-phosphocreatine kinase, activation of Rap1B induced by stimulation of either PAR-1 or PAR-4 was totally inhibited. By contrast, thrombin-induced activation of Rap1B was only minimally affected by neutralization of secreted ADP. Concomitant stimulation of both PAR-1 and PAR-4 in the presence of ADP scavengers still resulted in a strongly reduced activation of Rap1B. A similar effect was also observed upon blockade of the P2Y12 receptor for ADP, as well as in P2Y12 receptor-deficient human platelets, but not after blockade of the P2Y1 receptor. Activation of Rap1B induced by thrombin was not affected by preincubation of platelets with the anti-GPIbalpha monoclonal antibody AK2 in the absence of ADP scavengers or a P2Y12 antagonist but was totally abolished when secreted ADP was neutralized or after blockade of the P2Y12 receptor. Similarly, cleavage of the extracellular portion of GPIbalpha by the cobra venom mocarhagin totally prevented Rap1B activation induced by thrombin in the presence of apyrase and in P2Y12 receptor-deficient platelets. By contrast, inhibition of MAP kinases or p160ROCK, which have been shown to be activated upon thrombin binding to GPIb-IX-V, did not affect agonist-induced activation of Rap1B in the presence of ADP scavengers. These results indicate that although both PAR-1 and PAR-4 signal Rap1B activation, the ability of thrombin to activate this GTPase independently of secreted ADP involves costimulation of both receptors as well as binding to GPIb-IX-V.

A Selective Role for Phosphatidylinositol 3,4,5-Trisphosphate in the Gi-dependent Activation of Platelet Rap1B

The small GTP-binding protein Rap1B is activated in human platelets upon stimulation of a G(i)-dependent signaling pathway. In this work, we found that inhibition of platelet adenylyl cyclase by dideoxyadenosine or SQ22536 did not cause activation of Rap1B and did not restore Rap1B activation in platelets stimulated by cross-linking of Fcgamma receptor IIA (FcgammaRIIA) in the presence of ADP scavengers. Moreover, elevation of the intracellular cAMP concentration did not impair the G(i)-dependent activation of Rap1B. Two unrelated inhibitors of phosphatidylinositol 3-kinase (PI3K), wortmannin and LY294002, totally prevented Rap1B activation in platelets stimulated by cross-linking of FcgammaRIIA, by stimulation of the P2Y(12) receptor for ADP, or by epinephrine. However, in platelets from PI3Kgamma-deficient mice, both ADP and epinephrine were still able to normally stimulate Rap1B activation through a PI3K-dependent mechanism, suggesting the involvement of a different isoform of the enzyme. Moreover, the lack of PI3Kgamma did not prevent the ability of epinephrine to potentiate platelet aggregation through a G(i)-dependent pathway. The inhibitory effect of wortmannin on Rap1B activation was overcome by addition of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), but not PtdIns(3,4)P(2), although both lipids were found to support phosphorylation of Akt. Moreover, PtdIns(3,4,5)P(3) was able to relieve the inhibitory effect of apyrase on FcgammaRIIA-mediated platelet aggregation. We conclude that stimulation of a G(i)-dependent signaling pathway causes activation of the small GTPase Rap1B through the action of the PI3K product PtdIns(3,4,5)P(3), but not PtdIns(3,4)P(2), and that this process may contribute to potentiation of platelet aggregation.

Nitric Oxide Stimulates the Phosphorylation of rap1b in Human Platelets and Acts Synergistically with Iloprost

Phosphorylation of rap1b in human platelets correlates with both an upward shift of the protein on sodium dodecyl sulfate polyacrylamide gels and the translocation of the phosphorylated protein to the cytosolic fraction of platelets. We reported that this phenomenon occurs in platelets in response to agents that stimulate adenylate cyclase and thereby activate the cyclic AMP-dependent protein kinase. We now have evidence that phosphorylation of rap1b in platelets is also induced by nitric oxide generating compounds through stimulation of guanylate cyclase and activation of the cyclic GMP-dependent protein kinase. We observed time-dependent phosphorylation of rap1b and dose-dependent inhibition of collagen-stimulated aggregation in washed platelets incubated with S-nitroso serum albumin. In the presence of a combination of iloprost and 3-morpholino-sydnonimine, when both PKA and PKG are activated, phosphorylation of rap1b increased synergistically to a level three times higher than the sum of their individual actions.

Platelet rap1B phosphorylation is a sensitive marker for the action of cyclic AMP- and cyclic GMP-increasing platelet inhibitors and vasodilators.

Rap1B, a ras-like protein expressed in high concentrations in human platelets, serves as a substrate for protein kinase A (PKA) and, eventually, protein kinase G (PKG). We measured rap1B phosphorylation by autoradiography of 32P-labeled proteins in platelets prelabeled with [32P]-orthophosphate. Platelets coincubated with histamine-stimulated human umbilical vein endothelial cells (EC) showed increased phosphorylation of the 50-Kd vasodilator-stimulated phosphoprotein (VASP) of 2.6 +/- 0.5-fold maximally and of rap1B of 17.5 +/- 7.1-fold maximally (mean +/- SE, n = 4). Incubation of platelets with prostacyclin (PGI2), the PGI2-analogue iloprost (ILO), the nitric oxide (NO) donors SIN-1 or sodium nitroprusside (SNP) showed greater concentration-dependent phosphorylation of rap1B than of VASP. Phosphorylation of rap1B had a slow time course and was irreversible in contrast to that of VASP, which was rapid and reversible. Phosphorylation of rap1B was dependent on an increase of platelet cyclic AMP and/or cyclic GMP. Very small concentrations of ILO (50 pM), PGI2 (1 nM), and SIN-1 (100 nM) increased rap1B phosphorylation. Rap1B phosphorylation could also be detected by Western blot after incubation of platelet-rich plasma (PRP) with ILO or SIN-1. Measurement of platelet rap1B phosphorylation is a novel tool that allows monitoring of the action of labile (PGI2, NO) and more stable (ILO, SIN-1, SNP) platelet inhibitors and vasodilators that increase intracellular cyclic AMP and cyclic GMP. Determination of rap1B phosphorylation by Western blot opens new possibilities of measuring platelet-EC interactions in clinical studies and of monitoring the action of systemically applied PGI2 analogues and nitrovasodilators in pharmacologic studies.

Synergistic phosphorylation of platelet rap1B by SIN-1 and iloprost

Human platelets suspended in plasma or buffer were incubated with low concentrations of the nitric oxide (NO)-donor 3-morpholino-sydnonime (SIN-1; 100 nM to 1 μM) and the stable prostacyclin analogue iloprost (50 or 100 pM) and analyzed for cyclic nucleotide levels and protein phosphorylation. SIN-1 and iloprost synergistically stimulated the phosphorylation of rap1B and the 50 kDa vasodilator-stimulated phosphoprotein. SIN-1 stimulated platelet cyclic GMP and cAMP-levels and enhanced the increase in cyclic AMP elicited by iloprost. It was found that the mechanism underlying the synergistic phosphorylation of the 50 kDa protein and rap1B was different: synergistic phosphorylation of the 50 kDA protein seemed to be mediated by activation of both protein kinases A and G, whereas the synergistic rap1B phosphorylation could be attributed entirely to activation of protein kinase A. Measurement of rap1B phosphorylation might be a useful tool to monitor the action of systemically applied prostacyclin-analogues and nitrovasodilators in pharmacological studies.

Ultrastructural localization of the small GTP-binding protein Rap1 in human platelets and megakaryocytes.

Several functions have been proposed for Rap1B in human platelets, including the regulation of phospholipase (PL) C gamma and Ca2+ ATPase. However, its localization is largely unknown. In the present study we have investigated the subcellular distribution of Rap1 by immunocytochemical techniques using affinity purified polyclonal antibodies raised against residues 121-137 common to the 95% homologous Rap1A and Rap1B proteins. By immunofluorescence, a positive labelling was obtained on intact resting platelets and was abolished after adsorption of the antibodies with the control peptide. Immunoelectron microscopy was then used to further define the subcellular localization of Rap1B in platelets and megakaryocytes (MK). In resting cells, immunolabelling for Rap1B was associated with the plasma membrane, mostly at its inner face, and lined the membrane of the open canalicular system (OCS). Some labelling was also found outlining the alpha-granules, identified as such by a double labelling with an anti-GPIIb-IIIa. On thrombasthenic platelets the same localization was observed. When platelets were stimulated by thrombin, immunolabelling for Rap1B was redistributed to the zones of fusion of the granules with the OCS, and to the plasma membrane with a higher concentration on pseudopods. Human MK expressed Rap1 and the staining revealed the association of the protein with the demarcation membranes and alpha-granules. This study presents a first approach to the localization of a small GTP binding-protein Rap1B in whole platelets and MK, and shows its association with both the plasma and OCS membranes, as well as with the alpha-granule membranes.

Correlated expression of the 97 kDa sarcoendoplasmic reticulum Ca2+-ATPase and Rap1B in platelets and various cell lines

Evidence has accumulated that cyclic AMP (cAMP)-induced phosphorylation of a Ras-related protein (Rap1) regulates platelet Ca2+ transport. As this transport was recently found to be controlled by two isoforms of sarcoendoplasmic reticulum Ca(2+)-ATPase (SERCA), the 100 kDa SERCA2b and the newly identified 97 kDa SERCA, we attempted to establish which isoform is involved in this regulation. For this purpose, we studied the expression and regulation of both the SERCA and Rap1 isoforms in platelets, haemopoietic cells and various cancer cell lines. SERCA2b was shown to be equally expressed in all the cell lines tested, as determined by detection of its phosphoenzyme formation and by Western blotting using an isoform-specific antibody. In contrast, the expression of the 97 kDa SERCA, studied by the same methods, varied from total absence in the cancer cells to high levels in the megakaryocytic cell lines. With regard to the potential regulatory Rap1 proteins, Western blotting showed different expression of total Rap1 isoforms among the cell lineages, thus ruling out any possible relationship between Rap1 and SERCA2b. However, the expression of Rap1 proteins correlated with that of the 97 kDa SERCA isoform. More refined analysis of the rap1A and rap1B isoforms by reverse transcription PCR and by determining cAMP-induced phosphorylation of Rap1B, i.e. its functional mechanism, confirmed the correlation between Rap1B and the 97 kDa SERCA expression. This relationship was also established by the concerted up-regulation of these two proteins demonstrated in the pathological model of platelets from hypertensive rats. It is concluded that the expressions of 97 KDa SERCA and Rap1B are related, suggesting that regulation of the platelet Ca(2+)-ATPase system by cAMP-induced phosphorylation of Rap1B specifically involves the 97 kDa SERCA.