Should postoperative haemoglobin and potassium levels be checked routinely following blood-conserving primary total joint arthroplasty?

AIMS: Despite declining frequency of blood transfusion and electrolyte supplementation following total joint arthroplasty, postoperative blood analyses are still routinely ordered for these patients. This study aimed to determine the rate of blood transfusion and electrolyte restoration in arthroplasty patients treated with a perioperative blood conservation protocol and to identify risk factors that would predict the need for transfusion and electrolyte supplementation. PATIENTS AND METHODS: Patients undergoing primary total joint arthroplasty of the hip or knee between July 2016 and February 2017 at a single institution were included in the study. Standard preoperative and postoperative laboratory data were collected and reviewed retrospectively. A uniform blood conservation programme was implemented for all patients. Need for blood transfusion or potassium supplementation was determined through a coordinated decision by the care team. Rates of transfusion and supplementation were observed, and patient risk factors were noted. RESULTS: The overall rate of blood transfusion was 1.06% in the study population of 1132 total joint arthroplasties performed in 1023 patients. Of the 12 patients requiring transfusion, 11 were female, ten occurred in patients undergoing total hip arthroplasty, and all 12 patients had a preoperative haemoglobin level less than 130 g/l. Operative duration and surgical blood loss were significantly greater in those patients requiring blood transfusion. Nearly all patients requiring transfusion had a history of, or risk factors for, cardiovascular disease. Potassium supplementation was required in 15.5% of the study cohort; 72% of these patients receiving potassium presented with a potassium level less than 4 mmol/l during preoperative testing, while the remaining 28% had a past medical history of either significant anaemia, cardiopulmonary, cardiovascular, or renal diseases that had required substantial medical management. CONCLUSION: A consistent blood-conserving perioperative strategy effectively minimized need for blood transfusion in total joint arthroplasty patients below previously reported rates in the literature. We suggest that postoperative full blood counts and basic metabolic panels should not routinely be ordered in these patients unless their preoperative haemoglobin and potassium is below 130 g/dl or 4 mmol/l respectively, and they have medical comorbidities.

Pivotal role of paracrine effects in stem cell therapies in regenerative medicine: can we translate stem cell-secreted paracrine factors and microvesicles into better therapeutic strategies?

Although regenerative medicine is searching for pluripotent stem cells that could be employed for therapy, various types of more differentiated adult stem and progenitor cells are in meantime being employed in clinical trials to regenerate damaged organs (for example, heart, kidney or neural tissues). It is striking that, for a variety of these cells, the currently observed final outcomes of cellular therapies are often similar. This fact and the lack of convincing documentation for donor-recipient chimerism in treated tissues in most of the studies indicates that a mechanism other than transdifferentiation of cells infused systemically into peripheral blood or injected directly into damaged organs may have an important role. In this review, we will discuss the role of (i) growth factors, cytokines, chemokines and bioactive lipids and (ii) microvesicles (MVs) released from cells employed as cellular therapeutics in regenerative medicine. In particular, stem cells are a rich source of these soluble factors and MVs released from their surface may deliver RNA and microRNA into damaged organs. Based on these phenomena, we suggest that paracrine effects make major contributions in most of the currently reported positive results in clinical trials employing adult stem cells. We will also present possibilities for how these paracrine mechanisms could be exploited in regenerative medicine to achieve better therapeutic outcomes. This approach may yield critical improvements in current cell therapies before true pluripotent stem cells isolated in sufficient quantities from adult tissues and successfully expanded ex vivo will be employed in the clinic.

Two subpopulations of thrombin-activated platelets differ in their binding of the components of the intrinsic factor X-activating complex.

Binding of fluorescein-labeled coagulation factors IXa, VIII, X, and allophycocyanin-labeled annexin V to thrombin-activated platelets was studied using flow cytometry. Upon activation, two platelet subpopulations were detected, which differed by 1-2 orders of magnitude in the binding of the coagulation factors and by 2-3 orders of magnitude in the binding of annexin V. The percentage of the high-binding platelets increased dose dependently of thrombin concentration. At 100 nm of thrombin, platelets with elevated binding capability constituted approximately 4% of total platelets and were responsible for the binding of approximately 50% of the total bound factor. Binding of factors to the high-binding subpopulation was calcium-dependent and specific as evidenced by experiments in the presence of excess unlabeled factor. The percentage of the high-binding platelets was not affected by echistatin, a potent aggregation inhibitor, confirming that the high-binding platelets were not platelet aggregates. Despite the difference in the coagulation factors binding, the subpopulations were indistinguishable by the expression of general platelet marker CD42b and activation markers PAC1 (an epitope of glycoprotein IIb/IIIa) and CD62P (P-selectin). Dual-labeling binding studies involving coagulation factors (IXa, VIII, or X) and annexin V demonstrated that the high-binding platelet subpopulation was identical for all coagulation factors and for annexin V. The high-binding subpopulation had lower mean forward and side scatters compared with the low-binding subpopulation ( approximately 80% and approximately 60%, respectively). In its turn, the high-binding subpopulation was not homogeneous and included two subpopulations with different scatter values. We conclude that activation by thrombin induces the formation of two distinct subpopulations of platelets different in their binding of the components of the intrinsic fX-activating complex, which may have certain physiological or pathological significance.

Both the high affinity thrombin receptor (GPIb-IX-V) and GPIIb/IIIa are implicated in expression of thrombin-induced platelet procoagulant activity.

Platelets activated by alpha-thrombin express surface procoagulant activity (PCA) that accelerates the conversion of prothrombin to alpha-thrombin. Following activation with 10 nM alpha-thrombin, the PCA of normal platelets was approximately five-fold higher than that of Bernard-Soulier platelets (lacking GPIb). Normal platelet PCA was inhibited approximately 50% by activation in the presence of the anti-GPIb MoAbs LJIb10 or TM60. Moreover, normal platelet PCA was completely abrogated in the presence of a combination of both LJIb10 and c7E3, a MoAb directed against alphaIIbbeta3 (GPIIb/IIIa). In contrast. PCA expressed by Bernard Soulier or Glanzmann platelets was not inhibited by either LJIb10 or c7E3 MoAb. The platelet activating peptide SFLLRN at 10 microM, a concentration which fully activates platelet aggregation and Ca2+ mobilization, generated PCA activity one fifth of that generated by alpha-thrombin at 10 nM but anti-PAR1 antibodies did not affect thrombin-induced PCA expression. These results demonstrate that GPIb mediates, at least in part, the thrombin-induced activation of platelets that leads to PCA, and that alphaIIbbeta3 is also involved in PCA generation, but these results do not support a major role for PAR1 in this activation.

Novel structurally altered P(2X1) receptor is preferentially activated by adenosine diphosphate in platelets and megakaryocytic cells.

Experimental and clinical data suggest the presence of multiple types of adenosine diphosphate (ADP) receptors, one coupled to ligand-gated cation channels (P(2X)) and others coupled to G-protein-coupled (P(2Y)) receptors. This report identifies cDNA for a structurally altered P(2X1)-like receptor in megakaryocytic cell lines (Dami and CMK 11-5) and platelets that, when transfected into nonresponsive 1321 cells, confers a specific sensitivity to ADP with the pharmacologic rank order of ADP > > ATP > > > alpha,beta-methylene-ATP as measured by Ca(++) influx. This receptor (P(2X1del)) contains a deletion of 17 amino acids (PALLREAENFTLFIKNS) that includes an NFT consensus sequence for N-linked glycosylation. Glycosylated forms of the P(2X1del) and P(2X1wt) receptors were indistinguishable electrophoretically by Western blot or by immunoprecipitation using available antihuman and antirat antibodies. These results indicate that the expression of the P(2X1del) receptor results in an influx of Ca(++) induced by ADP. Expression of P(2X1del) receptor homomeric subunits is sufficient to express a receptor preferentially activated by ADP and suggests that this altered form, alone or in combination with P(2X1wt) receptors, is a component of an ADP-activated ion channel.

Activation of factor VIII by thrombin increases its affinity for binding to synthetic phospholipid membranes and activated platelets.

Membrane-bound thrombin-activated factor VIII (fVIIIa) functions as a cofactor for factor IXa in the factor Xase complex. We found that binding of heterotrimeric fVIIIa (A1.A2.A3-C1-C2) to synthetic vesicles with a physiologic content of 4% phosphatidylserine (PS), 76% phosphatidylcholine, and 20% phosphatidylethanolamine occurs with a 10-fold higher affinity than that of factor VIII (fVIII). The increased affinity of fVIIIa for PS-containing membranes resulted from the reduced rate of fVIIIa dissociation from the vesicles compared with that of fVIII. Similar affinities of A3-C1-C2, A1.A2. A3-C1-C2, and A3-C1-C2.heavy chain for interaction with PS-containing membranes demonstrate that removal of the light chain (LCh) acidic region by thrombin is responsible for these increased affinities of fVIIIa and its derivatives. Similar kinetic parameters of fVIII and its LCh and C2 domain for binding to PS-containing membranes and to activated platelets indicated that the C2 domain is entirely responsible for the interaction of fVIII with membranes. We conclude that the increased fVIIIa affinity for PS-containing membranes is a result of conformational change(s) within the C2 domain upon removal of the acidic region of the LCh. This conclusion is based on the finding that binding of the monoclonal antibody ESH8 to the C2 domain, which is known to prevent this conformational transition, resulted in fVIIIa binding to PS/phosphatidylcholine/phosphatidylethanolamine vesicles (4/76/20) with a lower affinity similar to that of fVIII. In addition, stabilization of the low affinity binding conformation of the C2 domain of fVIIIa by this antibody led to an inhibition of the fVIIIa activity in the factor X activation complex.

Proteolysis of platelet cortactin by calpain.

Cortactin, a substrate of pp60(c-)src and a potent filamentous actin binding and cross-linking protein, is abundant in circulating platelets. After stimulation of platelet aggregation with collagen, cortactin undergoes a dramatic increase in tyrosine phosphorylation followed by a rapid degradation. The cleavage of platelet cortactin was detected in lysates prepared using either Triton-containing buffer or SDS-sample buffer. However, the degradation of cortactin was not observed in platelets derived from a Glanzmann's patient, who lacked functional integrin alphaIIbbeta3 (GPIIb-IIIa). In addition, the proteolysis of cortactin was abolished by treating platelets before but not after collagen stimulation with EGTA or calpeptin. Furthermore, recombinant cortactin was digested by mu-calpain in vitro in a dose-dependent manner, indicating that cortactin is a substrate for calpain. We also observed that the calpain-mediated digestion in vitro is dependent on the presence of a sequence containing a proline-rich region and multiple tyrosine residues that are phosphorylated by pp60(c-)src. Tyrosine phosphorylation by pp60(c-)src up-regulates the activity of calpain toward cortactin. Our data suggest that the calpain-mediated proteolysis of tyrosine-phosphorylated cortactin may provide a mechanism to remodel irreversibly the cytoskeleton in response to platelet agonists.

Functional expression of a P2T ADP receptor in Xenopus oocytes injected with megakaryocyte (CMK 11-5) RNA.

Since the P2T purinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca2+ levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca2+ mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+ mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P2Y receptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-alpha-S are active as agonists and ATP and ATP-alpha-S are inactive as agonists but act as antagonists. [3H]ADP and 35S-ATP-alpha-S bound to CMK 11-5 cells at a high-affinity site (Kd1 and Ki1, 262 and 125 nmol/L, respectively) and a low-affinity site (Kd2 and Ki2, 10,100 and 5400 nmol/L, respectively) with 2 x 10(6) to 6 x 10(6) sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-alpha-S with affinities in a rank order similar to that found for platelets (ATP-alpha-S approximately ATP approximately ADP > or = ADP-beta-S approximately adenosine), suggesting the presence of a P2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with 35S-ATP-alpha-S resulted in the labeling of the alpha-subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected into Xenopus oocytes, only ADP and ADP-alpha-S stimulated 45Ca2+ efflux, which was not observed with ATP, 2-methylthio-ATP, alpha, beta-methylene-ATP, ATP-gamma-S, ATP-alpha-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-alpha-S but not adenosine blocked the 45Ca2+ response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P1, P2Y, P2X, and P2U agonists was expressed in functional form on Xenopus oocytes.

Contributions of glycoprotein Ib and the seven transmembrane domain receptor to increases in platelet cytoplasmic [Ca2+] induced by alpha-thrombin.

The individual contributions of glycoprotein Ib (GPIb) and the seven transmembrane domain receptor (STDR) to increases in platelet [Ca2+]i induced by alpha-thrombin or the tethered ligand peptide (TLP; SFLLRNPNDKYEPF) have been determined in control platelets, in platelets where the thrombin binding site on GPIb was blocked with the monoclonal antibodies TM60 and LJ-Ib10, in platelets where access of thrombin to the STDR was blocked by polyclonal antipeptide antibodies, and in Bernard-Soulier platelets which constitutively lack GPIb. Curve-fitting analyses (LIGAND) showed that binding of PPACK-thrombin and alpha-thrombin to the moderate-affinity site was not detected in the best-fit model in the presence of anti-STDR antibodies although with alpha-thrombin there was also decreased binding at the high-affinity site. Conversely, TM60 blocked binding of alpha-thrombin to the high-affinity site but also decreased binding at the moderate affinity site. Separately, either TM60 or anti-TNA (150 micrograms/mL) reduced thrombin (0.5 nM)-induced elevations in [Ca2+]i to 50% of control values, but Ca2+ elevations were essentially abrogated (4.2 +/- 5%) when the two were added in combination. [Ca2+]i dose-response curves for alpha-thrombin were curvilinear and were only 50% of controls in the presence of anti-GPIb or anti-STDR antibodies at up to 10 nM alpha-thrombin, with their greatest sensitivity being below 2 nM. With Bernard-Soulier platelets, changes in [Ca2+]i were not detectable at < or = 0.5 nM alpha-thrombin but were also 50% of controls at 5-10 nM alpha-thrombin. [Ca2+]i responses to TLP (1-100 microM) of antibody-blocked platelets were identical to those of controls whereas responses were approximately 50% of controls in Bernard-Soulier platelets. The rate of increase in [Ca2+]i in controls was twice that seen in antibody-blocked platelets and about 5-fold greater than in Bernard-Soulier platelets. These results demonstrate that both GPIb and the STDR are required to ensure the optimal rate and extent of platelet activation over a range of alpha-thrombin concentrations (0.3-10 nM) and that the STDR corresponds to the previously described moderate-affinity thrombin receptor.

Differentiation of the two forms of GPIb functioning as receptors for alpha-thrombin and von Willebrand factor: Ca2+ responses of protease-treated human platelets activated with alpha-thrombin and the tethered ligand peptide.

Previous results have shown that both GPIb and the seven transmembrane domain receptor (STDR) are required for optimal thrombin-induced platelet activation (Greco et al., 1996). Limited degradation (approximately 10%) of GPIb and the STDR by elastase reduced the Ca2+ response to 0.5 nM alpha-thrombin by only 10% whereas Serratia marcescens metalloprotease reduced the Ca2+ response by 80% and fully abrogated high-affinity thrombin binding and aggregation. vWF/ristocetin-induced agglutination was only slightly reduced (20%) while Ca2+ and aggregation response to higher thrombin concentrations were retained. At increasing elastase and Serratia protease concentrations, degradation of the STDR proceeded from the amino-terminal domain, but Ca2+ responses to the tethered ligand peptide SFLLRNPNDKYEPF were not affected by either protease. These results show that both putative thrombin receptors are susceptible to protease degradation and suggest that Serratia protease is able to differentiate the GPIb-mediated events associated with thrombin activation from those associated with ristocetin-induced agglutination.

Adenine nucleotide binding and photoincorporation in Glanzmann's thrombasthenia platelets.

Adenosine 5'-(1-thiotriphosphate) (ATP alpha S) binds to about 25,000 high affinity sites in platelets (Kd approximately 3 nM), competes fully in inhibiting the binding of ADP and, despite the absence of a specific photoactivatable substituent, is directly photoincorporated into a specific 18 kDa domain beginning at Tyr-198 in the alpha chain of glycoprotein IIb (GPIIb alpha) following ultraviolet irradiation of fresh unfixed platelets (Greco et al. (1991) J. Biol. Chem. 266, 13627-13633). 8-azido ATP has now been shown to have similar binding parameters (Kd 8 nM, 20,000 sites/platelet) but, in this case, photoincorporation occurred equally in GPIIb and GPIIIa. To determine the possible function of GPIIb alpha in ADP-induced activation, platelets were isolated from two Glanzmann's thrombasthenia patients whose platelets contain approximately 6% of normal levels of GPIIb. ADP and ATP alpha S bound to intact, formaldehyde-fixed Glanzmann's platelets at high affinity sites with dissociation constants of approximately 30 nM and approximately 2 nM, respectively. Both nucleotides also bound to low affinity sites with dissociation constants of approximately 2 microM: these values are similar to those obtained with control platelets. ATP alpha S antagonized the shape ADP-induced shape change response of Glanzmann's platelets (EC50 5 microM) indicating that it bound to the P2T (ADP) receptor. However, photoincorporation was low (approximately 7% of control) similar to their content of GPIIb alpha. These results show that ADP binding and photoincorporation are occurring at different sites on the platelet surface but suggest that the ADP binding site may be located in proximity to GPIIb alpha.

Thrombin binding to platelets defines functional receptors: inhibition of thrombin-induced platelet activation by catalytically-inactivated thrombin.

It has been widely questioned as to whether the observed binding of a-thrombin to intact platelets defines receptors coupled to signal transduction or merely thrombin binding sites. We have now shown that at α-thrombin concentrations sufficient to induce a full shape change response without aggregation (0.1 nM), PPACK-thrombin (that is, α-thrombin treated with the irreversible active site inhibitor D-phenylalanyl-L-prolyl-L-arginine chloromethylketone) dose-dependently inhibits platelet shape change (IC(50)∼70 nM), the concomitant increases in [Ca(2+)Ii (IC(50)∼75 nM) and ATP secretion (IC(50)∼50 nM). Since PPACK-thrombin competes fully in the binding of a-thrombin to high, moderate and low affinity sites on intact platelets, these results show that this binding defines functional receptors coupled to platelet activation.

Platelet activation and inhibition of malarial cytoadherence by the anti-CD36 IgM monoclonal antibody NL07.

The surface glycoprotein CD36 (GPIV) is known to mediate the adhesion of Plasmodium falciparum malaria-infected red blood cells and to be a receptor for extracellular matrix proteins such as collagen and thrombospondin. The murine monoclonal IgM antibody NL07, which is specific for CD36, has now been shown to also be a potent inhibitor of the adhesion of P falciparum malaria-infected red blood cells to C32 melanoma cells. Treatment of platelets with NL07 monoclonal antibody resulted in rapid degranulation, release of ATP and serotonin, increase in [Ca2+]i, and tyrosine phosphorylation of a substrate protein of 130 kD. In about one-half of the experiments, activation with NL07 resulted in the formation of small aggregates of 10 to 30 platelets, whereas in the other half of the experiments, large aggregates were seen similar to those induced by adenosine diphosphate (ADP) and these large aggregates could be converted to the small aggregates by ATP alpha S or by AP-2 or other antibodies against GPIIb and/or IIIa. Microaggregates of 2 to 5 platelets were seen with Glanzmann's platelets that constitutively lack GPIIb/IIIa. Aggregate formation was not seen with heat-treated serum, in the presence of anti C1q antibodies, or when using C5-, C8-, or C9-deficient human sera. Although activation of platelets with purified complement components results in a slow morphologic change without aggregation, involvement of CD36 results in rapid complement-mediated activation leading to formation of small aggregates that is largely independent of GPIIb/IIIa and that, under certain circumstances, proceeds to the formation of large ADP-dependent aggregates.

Changes in cytoplasmic pH in platelets activated through the high- and moderate-affinity receptor pathways by highly purified human alpha-thrombin.

Platelets in plasma were loaded with the probe BCECF/AM, and changes in cytoplasmic pH levels induced by highly purified human alpha-thrombin (2900 NIH U/mg) were studied in washed platelets having high- and moderate-affinity receptors and in platelets from which the high-affinity alpha-thrombin receptor had been removed by treatment with Serratia marcescens protease. In intact platelets, cytoplasmic acidification reached a maximum within 2 minutes of -0.072 +/- 0.009 pH units at 0.3 nmol/L alpha-thrombin concentration (0.03 U/ml). Cytoplasmic pH values were higher at both lower and higher alpha-thrombin concentrations and were significantly (p = 0.018) higher at 2 nmol/L alpha-thrombin, which induced -0.037 +/- 0.013 pH units of acidification. Five nanomoles of alpha-thrombin, however, induced cytoplasmic alkalinization of +0.027 +/- 0.033 pH units. In platelets lacking the high-affinity receptor where there is a 10 to 20-fold reduction in sensitivity to alpha-thrombin, acidification reached a maximum of -0.175 +/- 0.033 pH units at 2 nmol/L alpha-thrombin, but alkalinization was observed at 5 nmol/L (+0.038 +/- 0.025) and 10 nmol/L (+0.042 +/- 0.007) alpha-thrombin. These results show that the transition from acidification to alkalinization occurs in the same range of alpha-thrombin concentrations (2 to 5 nmol/L) in both preparations, despite the rightward shift in sensitivity caused by the absence of the high-affinity receptor. However, the maximum acidification reached in control platelets (-0.037 pH units at 2 nmol/L) was much less than the value obtained in platelets lacking the high-affinity receptor (-0.175 pH units at 2 nmol/L alpha-thrombin).(ABSTRACT TRUNCATED AT 250 WORDS)

Phencyclidine binds to blood platelets with high affinity and specifically inhibits their activation by adrenaline.

The ion channel probe phencyclidine [1-(1-phenylcyclohexyl)piperidine; PCP] selectively inhibited aggregation, secretion and ultrastructural changes in platelets induced by adrenaline, but did not affect activation induced by other common platelet agonists such as alpha-thrombin, ADP, collagen or ionophore A23187. [3H]PCP bound to platelets with high affinity (Kd 134 +/- 33 nM; 3600 +/- 1020 sites/platelet), as did the thienyl analogue [3H]TCP (1-[1-(2-thienyl)cyclohexyl]piperidine). PCP binding to platelets was increased 3-4-fold in N-methylglucamine buffer in the absence of Na+ ions. Binding was unaffected by haloperidol and was only weakly inhibited (EC50 10-20 microM), without significant stereoselectivity by the two sets of stereoselective ligands, dexoxadrol/levoxadrol and (+)MK801/(-)MK801. Binding of PCP was not competed for by adrenaline or yohimbine. Only the high-affinity binding of [3H]PCP to platelets was blocked by prior treatment of the platelets with the covalent affinity probe Metaphit, and these platelets no longer aggregated in response to adrenaline although they responded normally to alpha-thrombin, ADP and collagen. These results suggest that platelets contain high-affinity receptors for PCP that can modulate adrenaline-induced platelet activation.

Production of platelet thromboxane A2 and arterial prostacyclin I2 from hypercholesterolemic rats.

The plasma cholesterol, plasma malonaldehyde (MDA), platelet thromboxane A2 (TXA2) and vascular prostacyclin (PGI2) were measured in male Sprague-Dawley rats fed diets supplemented with cholesterol (1%) and cholic acid (0.5%). For comparisons, measurements were made in rats fed normal diets. The concentration of cholesterol in the plasma of rats had reached a maximum in 1 week of feeding experimental diets. TXA2 production from collagen and thrombin stimulated platelets was significantly decreased in animals fed experimental diets for 1 week. The production of MDA in the plasma of animals fed experimental diets for 8 weeks was significantly lower compared to the animals fed normal diets. There was a small but significant reduction in the formation of PGI2 in rats fed experimental diets for 8 weeks. These data suggest that feeding cholesterol rich diets to rats alters the platelet membrane properties differently from human and rabbit. Furthermore, cholesterol feeding to rats had some damaging effect on the arterial PGI2 synthesis.

Low structural specificity for nucleoside triphosphates as antagonists of ADP-induced platelet activation.

Although the platelet ADP receptor is thought to exhibit a high degree of structural selectivity, adenosine 5'-O-(thiotriphosphate) (ATP alpha S) is a potent inhibitor of ADP-induced platelet activation and has been recently shown to bind with high affinity (Kd 3 +/- 0.1 nM) to formaldehyde-fixed platelets and to be photoincorporated into an 18-kDa fragment beginning at Tyr-198 of glycoprotein (GP) IIb alpha (Greco, N. J., Yamamoto, N., Jackson, B. W., Tandon, N. N., Moos, M., Jr., and Jamieson, G. A. (1991) J. Biol. Chem. 266, 13627-13633). Further studies have now shown that guanosine 5'-O-(thiotriphosphate) (GTP alpha S) also binds to high affinity sites (Kd 4.7 +/- 0.9 nM; 13,600 +/- 1,140 sites/platelet) and to low affinity sites (Kd 470 +/- 85 nM; 135,900 +/- 19,400 sites/platelet). Competition binding studies showed that all GTP alpha S binding sites were accessible to ADP and vice versa. The corresponding pyrimidine nucleotide cytidine 5'-O-(thiotriphosphate) (CTP alpha S) was found to be similarly effective in competing in the binding of ADP and both 5'-O-(thiotriphosphates) as well as uridine 5'-O-(thiotriphosphate) (UTP alpha S) were potent inhibitors of platelet shape change and aggregation. Ultraviolet irradiation of platelets in the presence of either [35S]GTP alpha S or [35S]UTP alpha S resulted in their specific incorporation into the alpha chain of GPIIb as previously shown with [35S]ATP alpha S. These results show that the structure of the nucleotide base has little influence on its ability to occupy the ADP-binding site on platelets, to function as an inhibitor of ADP-induced activation or to be photoincorporated into GPIIb alpha.

Adhesive functions of platelets lacking glycoprotein IV (CD36).

Glycoprotein IV (GPIV; CD36 or GPIIIb) is a cell surface glycoprotein that has been proposed as mediating a number of physiologically important processes such as the adhesion of platelets to thrombospondin (TSP) and collagen, the cytoadherence of Plasmodium falciparum-infected erythrocytes, and the TSP-dependent interaction of monocytes with platelets and macrophages. Because platelets of the Naka-negative phenotype have recently been shown to lack detectable GPIV, their availability offered the opportunity to test directly these hypotheses regarding its adhesive functions. It has been found that Naka-negative platelets and monocytes do not support cytoadherence of P falciparum-infected erythrocytes. Naka-negative platelets are deficient in the initial stages of their adhesion to fibrillar collagen and this defect is most marked under Mg(2+)-free conditions. Finally, the ability of Naka-negative platelets to bind TSP before or after activation is unimpaired as compared with normal controls. These results do not support a role for GPIV as the TSP receptor.