Fresh Human Platelets Research Articles

Freeze-dried platelets promote clot formation, attenuate endothelial cell permeability, and decrease pulmonary vascular leak in a murine model of hemorrhagic shock.

Hemorrhagic shock (HS) and trauma induce endothelial barrier compromise, inflammation, and aberrant clotting. We have shown that fresh human platelets (Plts) and Plt extracellular vesicles mitigate vascular leak in murine models of injury. Here, we investigate the potential of freeze-dried platelets (FDPlts) to attenuate pulmonary vascular permeability, decrease inflammation, and promote clotting in a murine model of HS. Human FDPlts were characterized using in vitro assays of Plt marker expression, aggregation, coagulation, and endothelial cell permeability. An intravital model of vascular injury in the mouse cremaster muscle was used to assess the ability of FDPlts to incorporate into clots. Mouse groups subjected to controlled hemorrhage for 90 minutes were (1) lactated Ringer solution (LR), (2) FDPlts, (3) fresh human Plts, (4) murine whole blood (WB), and (5) shams (only instrumented). Hemorrhagic shock mouse endpoints included coagulation, pulmonary vascular permeability, and lung injury. Freeze-dried Plts expressed Plt-specific markers and retained functionality similar to fresh Plts. In in vitro assays of Plt aggregation, differences were noted. In vivo, FDPlts and Plts were found to incorporate into clots in postcapillary venules in the mouse cremaster muscle. Hemorrhagic shock mice resuscitated with LR displayed increased pulmonary vascular permeability compared with sham (sham, 686.6 ± 359.7; shock-LR, 2,637 ± 954.7; p = 0.001), and treatment with FDPlts or WB attenuated permeability compared with shock: shock-FDPlts, 1,328 ± 462.6 (p = 0.05), and shock-WB, 1,024 ± 370.5 (p = 0.0108). However, human Plts (Days 1-3) did not attenuate vascular leak in HS mice compared with shock-LR (shock-Plts, 3,601 ± 1,581; p = 0.33). FDPlts contribute to clot formation similar to fresh human Plts. FDPlts also attenuated vascular permeability in vitro and in vivo. Mouse WB resuscitation but not fresh human Plts attenuated vascular permeability after HS. These data suggest that the effect of FDPlts may be a suitable alternative to fresh Plts in modulating hemostasis and the endotheliopathy associated with injury.

Cofilin-1 Activation in Stored Platelets

BackgroundPlatelets, harvested for transfusion, have a relatively short “shelf life” of 5 days. With increasing storage, platelets develop structural and functional changes called “platelet storage lesions”. These changes result not only in reduced hemostatic response to agonists but also in an accelerated clearance from the circulation in vivo. The precise mechanisms of clearance of platelets aged in vitro (or vivo) are not known. There is an increased exposure of the anionic phospholipid phosphatidylserine (PS) on the cell surface of stored platelets. PS is a well-known tag for clearance of cells from the circulation by the macrophage PS receptors. PS-expressing stored platelets are also rapidly cleared from the circulation, presumably through the similar mechanism. Targeting this pathway could improve the recovery of stored platelets following transfusion by inhibiting storage-induced PS exposureResults and DiscussionActin is a major PS binding proteins in platelets. In resting platelets, actin filaments are at the periphery and in close proximity to the membrane. We hypothesize that the interaction with actin filaments constrains the mobility of PS in the inner leaflet. We generated artificial bilamellar liposomes mimicking the intra (PS) or extra (PC) cellular phospholipid composition of the membrane bilayer. When these liposomes were incubated with platelet lysate, the most striking enrichment was seen in actin, which was increased 5-fold in PS-containing liposome compared to control PC liposomes. These data suggest that actin and actin-associated proteins may, in fact, be the major inner leaflet PS-binding proteins in resting platelets.Cortical actin retracts from the membrane with PS exposure in senescent platelets: We examined the morphological changes in resting fresh platelets, five-day-old platelets and ABT-737 (10 μM) treated platelets by immunofluorescence microscopy. Platelets immobilized on polylysine-coated plates were immunostained with FITC-lactadherin and Alexa- 647-phalloidin. As shown in Figure 1, resting platelets do not express PS (no or minimal FITC-lactadherin staining). Actin filaments (Alexa-647-phalloidin) are distributed in the subcortical region as a thin submembranous network. In 6-day old stored platelets, there is significant increase in platelet surface FITC-lactadherin staining. In addition, in most platelets actin filaments have retracted from the subcortical region towards the center of the platelet (compare figure 1 panels A and BBad phosphorylation is significantly diminished in stored platelets. Phosphorylated Bad is sequestered in the cytosol by binding to the scaffolding protein 14-3-3. Furthermore, phosphorylation of S118 inhibits apoptosis by preventing its association with Bcl-xL. Dephosphorylation of Bad may initiate intrinsic apoptosis by associating with Bcl-xL. We determined the phosphorylation status of Bad in stored platelets by Phosphoproteomic and WB analyses of stored and fresh human platelets ( Figure-2) Both methods showed a drastic decrease in Bad is phosphorylation at serine residues 99 and 118 in stored platelets.Activation of cofilin-1 by dephosphorylation in stored platelets. Like Bad, phosphorylation of cofilin-1 is also markedly decrease in stored platelets as shown by WB analyses (Figure-3). It is unclear how the apoptotic pathways lead to cofilin-1 activation (dephosphorylation). Caspases are known to activate slingshot phosphatase which could then activate cofilina1 by its dephosphorylation. LIM- kinases phosphorylate and, thus, inactivate cofilin-1 whereas slingshot phosphatases dephosphorylate and activate cofilin-1. Our data show that the Lim kinase inhibitor, as expected increased ABT-737-induced PS exposure on platelets (Figure 4A), whereas slingshot phosphatase inhibitor inhibited ABT-737-induced PS exposure at a 1-μM concentration (Figure 4B)ConclusionOur data show that activation of apoptotic pathways is responsible for activation of cofilin and subsequent PS exposure by actin reorganization in stored platelets. Targeting this pathway could improve the recovery of stored platelets following transfusion by inhibiting storage-induced PS exposure. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Reprint of “Ultrananocrystalline diamond integration with pyrolytic carbon components of mechanical heart valves”

In this report, an idea of integrating ultrananocrystalline diamond (UNCD) with pyrolytic carbon (PyC)-based mechanical heart valves, has been demonstrated. The report addresses the strategies to avoid graphitization and film delamination during the diamond coating. Raman and scratch tests showed that a UNCD film with high purity could adhere to the PyC substrate strongly. A thrombin generation study demonstrated an excellent biocompatibility of UNCD towards fresh human platelets. These results suggest that UNCD could be a good candidate of surface material for next generation heart valves and other implantable devices.

Ultrananocrystalline diamond integration with pyrolytic carbon components of mechanical heart valves

In this report, an idea of integrating ultrananocrystalline diamond (UNCD) with pyrolytic carbon (PyC)-based mechanical heart valves, has been demonstrated. The report addresses the strategies to avoid graphitization and film delamination during the diamond coating. Raman and scratch tests showed that a UNCD film with high purity could adhere to the PyC substrate strongly. A thrombin generation study demonstrated an excellent biocompatibility of UNCD towards fresh human platelets. These results suggest that UNCD could be a good candidate of surface material for next generation heart valves and other implantable devices.

Kindlin-3 Undergoes Endoproteolysis in Stored Platelets and After Platelet Stimulation

Abstract 1255Kindlins are a family of FERM domain proteins that are essential for inside-out integrin activation. In particular, kindlin-3 is required for the conversion of the major platelet integrin αIIbβ3 from its resting conformation to its active ligand binding conformation. Moreover, naturally-occurring kindlin-3 mutations result in the inherited disorder leukocyte adhesion deficiency III, one component of which is defective platelet function that mimics Glanzmann thrombasthenia. Despite the importance of kindlin-3 in initiating αIIbβ3 function in platelets, little is known about its regulation in resting platelets or its fate in activated platelets. To address these questions, we purified full-length kindlin-3 from outdated human platelets where it is present in substantial amounts and also developed a procedure to synthesize substantial amounts of recombinant kindlin-3 in SF9 cells. We found that in stored human platelets, kindlin-3 is cleaved into two fragments as a function of the time of storage. We also found that kindlin-3 is cleaved into identical fragments when fresh human platelets are stimulated with the thrombin receptor activating peptide (TRAP) for 5 minutes. To identify the site of kindlin-3 cleavage, as well as the responsible protease, we used a proteomics method in which an engineered peptide ligase, subtiligase, was used to selectively biotinylate the unblocked α amines of proteins obtained from platelet lysates (Mahrus et al, Cell 134:866–76, 2008). Biotinylated proteins were digested with trypsin and the resulting biotinylated peptide fragments were then captured using avidin agarose and identified using tandem mass spectrometry. Using this method, we identified the kindlin-3 peptide (G)SAPTDVLDSLTTIPELKDHL in lysates of stored platelets, thereby mapping the cleavage site to residues 335–336. We obtained identical results using proteins isolated from TRAP-stimulated platelets. Further, we were able to recapitulate these results in vitro using purified kindlin-3 and the calcium-activated protease calpain, implying that calpain is the responsible protease in vivo. Kindlin-3 is thought to initiate αIIbβ3 function by binding to the distal NITY motif in the β3 cytosolic tail in an interaction that also involves S752. Previously, we reported a model for αIIbβ3 regulation based on an NMR structure of the β3 cytosolic tail (Metcalf et al, PNAS 107:24775–83, 2010). In this structure, the NITY motif is located in a distal dynamic amphiphilic helix where the motif is transiently masked by interacting with the membrane. To validate this model, we have studied the interaction of both purified and recombinant kindlin-3 with the β3 tail using surface plasmon resonance (SPR). A peptide corresponding to β3 residues 719–762, encompassing the complete β3 tail, was immobilized on a CM5 chip and kindlin-3 was flowed over the chip surface. The resulting sensorgrams could then be fit to two binding events with dissociation constants of 2.2 nM and 2.8 μM. The biphasic behavior could have resulted from heterogeneity of the β3 tail on the chip surface or heterogeneity of the interaction between kindlin-3 and the carboxymethylated dextran. Similar SPR experiments measuring binding of the talin-1 FERM domain to the β3 tail also could be fit to two binding events with dissociation constants of 155 nM and 3.5 μM. Thus, under these experimental conditions, kindlin-3 binds approximately 70-fold more tightly to the β3 tail than does the talin-1 FERM domain. In summary, these studies demonstrate that kindlin-3 undergoes calpain-mediated endoproteolysis during platelet storage, an event that may contribute to the development of the platelet storage lesion. Kindlin-3 also undergoes an identical cleavage following platelet stimulation by agonists such as thrombin. Since high affinity binding of kindlin-3 to the β3 cytosolic tail is required for physiologic αIIbβ3 activation, it is possible that kindlin-3 cleavage attenuates αIIbβ3 activity. Disclosures:No relevant conflicts of interest to declare.

Effects of UV-irradiated titania nanoparticles on cell proliferation, cancer metastasis and promotion

The antitumor activity of irradiated Degussa P25 TiO 2 nanoparticles (anatase/rutile ratio of about 3:1; average diameters of the anatase and rutile particles estimated at 25 and 85 nm, respectively) was investigated in vitro: sarcoma cells killing, lipooxygenase enzymic action inhibition; and ex vivo: human platelets aggregation, in order to get insight into the possible effects of photoexcited titanium dioxide in cancer treatment. Irradiated and non-irradiated TiO 2 concentrations (varying from 0.8 μM to 1000 μM) did not show any toxicity when cultured with fibroblasts MRC-5. In vitro cultured sarcoma cells, derived and isolated from sarcoma tumors produced on experimental Wistar rats after their treatment with benzo-[a]-pyrene, were completely killed in the presence of TiO 2 (40 μg/ml). The antimetastatic capability of titanium oxide in haematogenous spread of cancer cells was also confirmed in tests involving fresh human platelets with a number of different stimulators, including sarcoma cells, platelet activated factor (PAF), diphosphoric adenosine (ADP) and arachidonic acid. Sarcoma cells-human platelets mixtures irradiated by UV-C in the presence of TiO 2 showed a significant decrease in aggregation, effect that becomes more pronounced by increasing the titania concentration. Platelet aggregation triggered by PAF and arachidonate addition was totally inhibited by photoexcited TiO 2, while partial inhibition of aggregation triggered by ADP was observed. Addition of TiO 2 followed by irradiation with UV-C also resulted in an important decrease of lipooxygenase enzyme's activity.

Expression of Tissue Factor and Initiation of Clotting by Human Platelets and Monocytes After Incubation With Porcine Endothelial Cells

Intravascular thrombosis remains a major barrier to successful pig-to-primate xenotransplantation. However, the precise factors initiating thrombosis are unknown. In this study, we investigated the contribution of recipient platelets and monocytes. Primary pig aortic endothelial cells (PAECs) were incubated with combinations of fresh or heat-inactivated human plasma, platelets, or monocytes, after which they were separated and analyzed individually by flow cytometry for tissue factor (TF) expression and for their ability to clot recalcified normal or factor-VII-deficient plasma. Procoagulant porcine TF was induced in PAECs only by fresh human plasma, and not by heat-inactivated plasma, platelets, or monocytes. In contrast, procoagulant human TF was induced on platelets and monocytes after incubation with PAEC, irrespective of whether the plasma was present or not. In addition, human platelets caused the shedding of procoagulant TF-expressing aggregates from PAEC. This work defines a cell-based in vitro assay system to address complex interactions among PAECs, human platelets, and monocytes. The induction of procoagulant TF on PAECs by fresh human plasma was most likely dependent on xenoreactive natural antibody and complement present in fresh human plasma. In contrast, the shedding of procoagulant platelet-PAEC aggregates, induced by human platelets, and the induction of procoagulant TF on human platelets and monocytes by PAEC, occurred independently of these factors. These results suggest that different mechanisms may contribute to the initiation of thrombosis after xenotransplantation, some of which may not be influenced by the further manipulation of the immune response against pig xenografts.

Treahlose Loaded, Freeze-Dried Rehydrated Human Platelets Maintain and Regulate Their Intracellular pH.

Maintenance of intracellular pH, a critical cellular function, is required for generation of proton gradients and platelet response to agonist. Previously, we have demonstrated that freeze-dried rehydrated platelets are able to respond to agonists generate a rise in intracellular calcium (Auh et al. 2004 Calcium mobilization in freeze-dried platelets. Cell Preservation Technology in press) and maintain normal membrane and protein secondary structure (Wolkers et al. 2002 Towards a clinical application of freeze-dried platelets. Cell Preservation Technology 1:175–188). As part of our ongoing studies of trehalose loaded freeze-dried rehydrated human platelets we examined their ability to maintain their intracellular pH. Platelet proton regulation is achieved through the Na-H exchanger (NHE1) which is dependent on the concentration of extracellular sodium. Freeze-dried and fresh control human platelets were loaded with the pH ratio dye bis-carboxyfluorescein acetomethyl ester (BCECF-AM), washed over a Sepharose 2B column and examined by fluorescence spectroscopy. Fresh and freeze-dried rehydrated human platelets maintained virtually identical resting intracellular pH, 7.273 +/− 0.015, and 7.270 +/− 0.034 respectively. Both cell types responded to increased extracellular sodium by increasing their pH in a virtually identical manner. The addition of 0.5U/ml thrombin (in the presence of 135 mM NaCl) resulted in an initial acidification and subsequent alkalinzation of both fresh and freeze-dried rehydrated cells. Prior to the addition of thrombin both cell populations had an [pH]i of 6.9, while after thrombin stimulation the pH rose to 7.012 for fresh cells and 7.001 for freeze-dried rehydrated cells. Thrombin stimulation in the absence of extracellular sodium resulted in a significant acidification of both cell populations to a final pH of 6.6 for fresh cells and 6.7 for freeze-dried rehydrated cells. Specific inhibition of the NHE1 transporter by 5-(N-methyl-N-isobutyl) amiloride (MIA) completely abolished the response of all cells to increasing concentrations of sodium. In the parallel control experiment both freeze-dried and fresh cells acidified to pH 6.2 and incubated with 135mM NaCl responded by generating a rise in intracellular pH to 7.1. These results demonstrate that freeze-dried rehydrated platelets are able to maintain normal pH homeostasis and respond to agonist in a specific manner. Studies funded by DARPA.

Dietary phytoestrogens and their synthetic structural analogues as calcium channel blockers in human platelets.

Phytoestrogens have been shown to inhibit platelet activation by blocking platelet calcium channels. This study examined the effect of several synthetic derivatives of trans-resveratrol, genistein, and daidzein on platelet free intracellular calcium ([Ca2+]i) elevation in thrombin-activated platelets and the possible mechanisms of this inhibitory effect. Studies were conducted on fresh human platelets from healthy volunteers. The fluorescent dye fura-2 was used to monitor [Ca2+]i in platelets. At 10 microM-resveratrol, triacetyl-trans-resveratrol, and trimethoxy-trans-resveratrol produced, respectively, 57 +/- 4%, 40 +/- 4%, and 21 +/- 1% inhibition; genistein, acetylgenistein, and dihydrogenistein produced 51 +/- 10%, 26 +/- 7%, and 16 +/- 2% inhibition, respectively; daidzein and diacetyldaidzein produced 56 +/- 5% and 45 +/- 10% inhibition of thrombin-induced [Ca2+]i elevation. The inhibitory effect was immediate and appeared to directly affect the calcium influx channels. Phytoestrogen action on [Ca2+]i did not cause alteration in nitric oxide signaling. Tyrosine phosphorylation was not involved in the inhibition of [Ca2+]i elevation by phytoestrogens, because the percent inhibition produced by the tyrosine kinase inhibitor genistein and its inactive analogue daidzein on thrombin-induced and thapsigargin-induced [Ca2+]i elevation was not significantly different for either compound at any concentration tested. Structure-activity relationship studies on this limited set of compounds reveal the requirements for the stilbene pharmacophore for the calcium-blocking activity.

Sequencing of Full-Length cDNA Encoding the α and β Subunits of Human Casein Kinase II from Human Platelets and Megakaryocytic Cells. Expression of the Casein Kinase IIα Intronless Gene in a Megakaryocytic Cell Line

Casein kinase II (CKII) is a ubiquitous protein kinase composed of two subunits, alpha and beta, that can use both ATP and GTP as phosphoryl donors. Two genes located on two separate chromosomes were identified for CKIIalpha: one on chromosome 20 band 13 with an approximate size of 20 kb and a second on chromosome 11 band 15.5-p15.4 that is the same size as the cDNA of locus 20 kb (1.2 kb) and does not contain any introns. The two genes differ in four amino acids. Recently, it has been demonstrated that a membrane-associated platelet-derived CKII phosphorylates coagulation factor Va. The mRNA encoding the platelet CKII was isolated from fresh human platelets, and the corresponding cDNAs encoding the alpha and beta subunits of human platelet CKII were produced and sequenced. The cDNA for platelet CKIIalpha was found to be 99.7% homologous to the CKIIalpha intronless gene, having the same characteristic amino acid residues at positions 128, 256, 287, and 351. However, the cDNA of platelet CKIIalpha has a different amino acid at position 236 (Arg --> His), which is not found in the intronless gene. The cDNA of the CKIIbeta subunit was completely identical with the sequence of the CKIIbeta subunit isolated from other tissues. Since platelets arise from megakaryocytes, mRNA was isolated from the megakaryocytic cell line MEG-01 and the cDNA for CKIIalpha was cloned and sequenced. The cDNA was found to be identical to the intronless gene found in platelets. We have also investigated the expression of the intronless gene in several other cell lines. Expression of the intronless gene was only found in cell line MEG-01. Our data demonstrate expression of the CKIIalpha intronless gene in megakaryocytes and platelets.

Novel treatment modalities: new platelet preparations and substitutes.

Keywords: platelets; platelet substitutes; platelet haemostatic function; blood substitutes; platelet function

Correlation of in vivo and in vitro functions of fresh and stored human platelets.

Long-term storage of human platelets has been hindered by the loss of function of the platelets stored under current protocols. Novel preservation methods have encouraged examination of platelet function of cells preserved by cooling and freezing. The function of the platelets was assessed by using both in vitro assays and an in vivo rabbit bleeding model. Human platelets were stored in the presence or absence of 2 microM: cytochalasin B and 80 microM: EGTA/AM at 4 degrees C for 14 days or by freezing in the presence or absence of 5 percent DMSO. After the storage period, the platelets were resuspended in normal saline and infused into rabbits. Platelet function was assessed in vivo in a kidney bleeding model and in vitro by platelet-induced clot retraction and by platelet aggregation. Platelets stored at either 4 degrees C or -145 degrees C exhibited shorter survival times in the rabbit circulation than did fresh platelets. Platelets cooled to 4 degrees C, in both the presence or absence of cytochalasin B and EGTA/AM treatment, or frozen in the absence of DMSO were not effective in halting bleeding. However, frozen DMSO-treated platelets were as effective as fresh platelets in stopping bleeding. In vitro assays showed that cooled platelets treated with cytochalasin B and egtazic acid/AM and frozen DMSO-treated platelets retained 30 to 40 percent platelet function, while the cooled and frozen control samples exhibited no platelet-induced clot retraction. With thrombin as the agonist, only frozen DMSO-treated platelets exhibited a tendency to aggregate, although at only 22 percent of the aggregation function of fresh platelets. It is possible to freeze platelets and retain in vivo efficacy if the cryopreservative DMSO is included in the preparation. In vitro responses were greatly reduced by all of the storage protocols, but it may not be necessary to retain 100 percent in vitro function to have a platelet substitute or storage product that functions satisfactorily in vivo.

Reduction of blood loss by infusion of human platelets in a rabbit kidney injury model

As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a kidney injury model was developed to assess the hemostatic properties of human platelets in normal and thrombocytopenic rabbits. New Zealand white rabbits were made thrombocytopenic by two consecutive injections of busulfan. Two weeks later, human platelets were transfused to animals whose reticuloendothelial systems were inhibited by the administration of ethyl palmitate. The left kidney was exposed and a slice excised from the anterior pole. The blood was contained in a parafilm boat and absorbed by preweighed gauze to assess blood loss. The percentage of human platelets transfused to the rabbit was determined by flow cytometry on blood collected from the cut site using anti-CD42a (marker for human platelets). The degree of activation of the human platelets was determined using anti-CD62a (marker specific for human p-selectin). Blood loss was similar in normal animals treated with saline alone (35.4 +/- 5.8 g; n = 4); ethyl palmitate and saline (42.5 +/- 5.7 g; n = 6, p = 0.4); or ethyl palmitate and fresh human platelets (45.7 +/- 7.9 g; n = 6, p = 0.3). Bleeding in thrombocytopenic rabbits infused with saline was increased (75.6 +/- 3.9 g; n = 7) as compared with nonthrombocytopenic animals. A significant reduction in blood loss was noted in thrombocytopenic rabbits given fresh human platelets (51.6 +/- 4.5 g; n = 6, p = 0.0023). Transfusion of human platelets to rabbits did not cause activation of the platelets. Furthermore, transfusion of thrombin-activated platelets (60-98% activated) to thrombocytopenic rabbits reduced blood loss (54 +/- 7.3 g; n = 7) to the same extent as fresh platelets. This is the first report describing a kidney injury model developed to assess the efficacy of fresh and activated human platelets in reducing blood loss in thrombocytopenic rabbits. This model could monitor the efficacy of human platelets prepared by various preservation protocols in suppressing bleeding in rabbits.

Survival of fresh human platelets in a rabbit model as traced by flow cytometry.

In the event of hemorrhage and blood loss, platelets play a vital role in the coagulation process. However, there are currently no acceptable protocols for long-term storage of platelets. As a first step toward testing the efficacy of stored platelets or platelet substitutes in vivo, a flow cytometric technique was developed to detect human platelets in rabbit blood. Human platelets were transfused to rabbits whose reticuloendothelial system was inhibited by the administration of ethyl palmitate. Because human and rabbit platelets display surface molecules with different epitopes, human platelets were selectively labeled with antibodies specific for glycoprotein IX (CD42a). As this antibody does not label rabbit platelets, it allows discrimination of human from rabbit platelets in samples of rabbit blood containing both types of platelets. Survival of human platelets in rabbits was monitored by flow cytometry and fluorescence microscopy in blood drawn at various times after the platelet transfusion. Fresh human platelets transfused to untreated control rabbits (n = 3) were removed from circulation within 10 minutes of the completion of the transfusion. Fresh platelets (1 day old) transfused to rabbits treated with ethyl palmitate (n = 5) survived for 24 hours with an average half-life of 8.6 hours. In contrast, 8-day-old platelets were cleared from the circulation sooner with an average half-life of 2.9 hours (n = 4). This report describes a rapid and efficient method of assessing the survival of human platelets in a rabbit model using flow cytometry. This technique will enable the monitoring in rabbits of human platelets prepared by various preservation protocols.

Purification and properties of a human plasma endogenous modulator for the platelet tricyclic binding/serotonin transport complex.

An endogenous modulator for the site labeled by [3H]imipramine which is putatively coupled to the serotonin transporter in human platelets was isolated and purified from plasma. Procedures included sequential chromatography on Cibacron blue-Sepharose 4B, concanavalin A-Sepharose 4B, Mono Q HR 10/10 anion exchange, DuPont GF-250 gel permeation and Mono S HR 5/5 cation exchange columns. The purified modulator is a protein of Mr 45,000 with a very acidic pK (less than 3) and sensitive to various proteinases but heat- and acid-stable. This protein inhibited [3H]imipramine binding to platelet membranes competitively (IC50 approximately 6 microM) and enhanced serotonin uptake in fresh human platelets (EC50 approximately 7 microM). Various physicochemical properties, including chromatographic, electrophoretic and immunological as well as amino acid composition analysis revealed that the isolated protein is most probably the human alpha 1-acid glycoprotein.

Study on the endogenous lectins of the human platelet plasma membrane

Plasma membraresisolated from human blood platelets and human intact platelets, either fresh or previously fixed, were investigated for their possible content in intrinsic sugar-binding proteins (lectins) which can specifically bind monosaccharides such as fucose, mannose, and galactose. Light and heavy fractions of platelet plasma membrane and intact platelets fixed with 2% formaldehyde and suspended in Ty ode's buffer pH 7.0 were incubated with either L-(1- 3H)-fucose, D-(1- 3H)-mannose or D-(1- 3H)-galactose. Intact fresh platelets were incubated either with L-(1- 3H)-fucose, D-(1- 3H)-mannose, D-(1- 3H)galactose, D-(1- 3H)-glucose and N-acetyl-D-(1- 3H)-glucosamine or with the same monosaccharides radiolabeled with ( 14C). Samples of radio-labeled plasma membranes and intact platelets were electrophoresed on polyacrylamide gels, and prepared for spectrometry. Results indicate that isolated human platelet plasma membranes contain sugar-binding proteins, especially for L-fucose, for which the binding values in the light fraction are about 4 times greater than for D-galactose. For the heavy fraction, the binding value for L-fucose is about 12 and 89 times higher than for D-mannose and D-galactose, respectively. Similar to plasma membrane fractions, fixed intact human platelets bind predominantly L-fucose, followed in decreasing binding values by D-mannose and D-galactose. Unlike these, fresh human platelets bind mainly D-galactose, followed in affinity values by D-glucose, L-fucose and D-mannose and to a lesser extent by N-acetyl-D-glucosamine. The binding of radiolabeled sugars to isolated platelet plasma membranes and to the previously fixed intact platelets suggests that, an endogenous lectin-like activity is expressed by the platelet membrane.

A human osteosarcoma cell line secretes a growth factor structurally related to a homodimer of PDGF A-chains.

Platelet-derived growth factor (PDGF), as purified from fresh human platelets, is a protein of relative molecular mass (Mr) 30,000 composed of two disulphide-linked subunit chains of similar size, named A and B (ref. 1). The dimer structure of PDGRF seems to be important for its biological effects, as reduction irreversibly inactivates the factor; it is not known, however, whether PDGF exists as a heterodimer or as a mixture of homodimers. Amino-acid sequence analysis has revealed that the A- and B-chains of human PDGF are related to each other, and that the B-chain is almost identical to part of the v-sis gene product of simian sarcoma virus (SSV). There is experimental evidence that a PDGF-like protein is indeed operational in SSV-induced transformation and the biologically active v-sis product is probably structurally similar to a putative dimer of PDGF B-chains. PDGF-like growth factors and/or a 4.2-kilobase (kb) c-sis transcript are present in several transformed mammalian cell lines and in certain nontransformed cells; cloned c-sis complementary DNA from human T cells transformed with human T-lymphotropic virus (HTLV) or from human endothelial cells contains the coding sequence for a putative PDGF B-chain precursor, but apparently lacks PDGF A-chain sequences. We have previously partially purified and characterized a PDGF-like growth factor from U-2 OS cells (osteosarcoma-derived growth factor, ODGF) and shown that this factor has structural, functional and immunological characteristics in common with PDGF. We describe here a procedure for the preparation of homogeneous ODGF, and provide evidence that this factor, which binds to the PDGF receptor, has a structure similar to a homodimer of PDGF A-chains.

血小板由来血管内皮細胞増殖因子（Ｖａｓｃｕｌａｒ Ｅｎｄｏｔｈｅｌｉａｌ Ｃｅｌｌ Ｐｒｏｌｉｆｅｒａｔｉｏｎ Ｆａｃｔｏｒ ｆｒｏｍ Ｐｌａｔｅｌｅｔｓ）の検討

Endothelial cell growth factor from platelets, designated as Vascular Endothelial cell Proliferation Factor (VEPF), was purified to an apparent homogeneity from fresh human platelets using eight steps, e. g.: QAE-Sephadex chromatography, ammonium sulfate precipitation, DEAE-Sepharose chromatography, chromatofocusing separation, Mono P chromatography and HPLC gel chromatography using Superose 12 and TSK 4000 columns. The half maximal stimulation was obtained at a concentration of less than 10ng/ml (less than 200pM). The highly purified material stimulated the growth of porcine aortic endothelial cells but was inactive against human foreskin fibroblasts.

Effect of phospholipase C (Bacillus cereus) on freshly isolated and 4-day-stored human platelets.

Phospholipase C (from Bacillus cereus) was used to study fresh and stored human platelets. Provided that the enzyme was inactivated before lipid extraction, no significant degradation of phospholipid in fresh cells was noted, even when platelets were activated or induced to change shape by ADP, collagen or thrombin. With platelets isolated from concentrates stored for transfusion for 4 days at 22 degrees C, membrane phospholipids were degraded by the enzyme to an extent depending on the pH in the platelet concentrate at day 4 of storage. The extent of phospholipid hydrolysis in platelets correlated well with the extent of release of lactate dehydrogenase during storage, with both being minimal for platelets from concentrates of final pH 6.5-6.9. Under non-lytic conditions, phosphatidylcholine was the phospholipid most degraded (40%), with no significant degradation of phosphatidylserine being detected. Storage does not seem to alter the distribution of phospholipids at the external leaflet of the plasma membrane.

Structural and biological characteristics of connective tissue activating peptide (CTAP-III), a major human platelet-derived growth factor.

Connective tissue activating peptides (CTAPs) extracted from leukocytes and platelets stimulate glycolysis and synthesis of glycosaminoglycan and DNA in cultured human connective tissue cells. CTAP-III, isolated from fresh or outdated human platelets, is a low molecular weight single-chain protein with an isoelectric point of 8.5 that markedly stimulates DNA synthesis and multiple aspects of glycosaminoglycan and proteoglycan metabolism. This report presents a definitive comparison of CTAP-III prepared by two methods [one designated (A), alternative] with similar platelet proteins described by others, beta-thromboglobulin (beta-TG) and low-affinity platelet factor 4 (LA-PF-4). CTAP-III, CTAP-III(A), LA-PF-4, and beta-TG have common antigenic determinants documented by immunoprecipitation and radioimmunoassay. CTAP-III, CTAP-III(A), and LA-PF-4 are biologically active in that they stimulate DNA and glycosaminoglycan synthesis by human synovial cells; beta-TG is inactive. Carboxyl-terminal digestion gave identical terminal sequences for CTAP-III, CTAP-III(A), and beta-TG. Amino-terminal sequence data indicate that CTAP-III and CTAP-III(A) (also LA-PF-4) are identical and differ from beta-TG only by an additional amino-terminal tetrapeptide (Asn-Leu-Ala-Lys-). The biologically active molecule, CTAP-III, may be proteolytically converted to its inactive degradation product (beta-TG) in the course of platelet aging, platelet storage, release from the platelets, or initiation of biological activity.