Immunologic Detection of Phosphatidylserine Externalization during Thrombin-Induced Platelet Activation

The antiphospholipid antibody syndrome is characterized by circulating antiphospholipid antibodies against cardiolipin (CL) and phosphatidylserine (PS) and clinically associated with a high risk of spontaneous thrombosis. Three monoclonal antibodies that differentiate between CL or PS were tested against resting and thrombin-activated platelets by flow cytometry. Each antibody reacted differently with CL and PS; 3SB9b reacted with PS, D11A4 reacted with CL, and BA3B5C4 reacted with both CL and PS. Activated platelets bound BA3B5C4 and 3SB9b, but not D11A4. The BA3B5C4-reactive epitope appeared earlier during activation than the epitope reactive with 3SB9b. These data suggest that antibodies against PS are reactive with activated platelets and that two immunoreactive forms of PS are sequentially expressed on platelets during activation.     

Platelet transfusions can induce transplantation tolerance

Recently we have shown that the induction of antibodies against the H-2 antigens after multiple platelet transfusions is due to leukocyte contamination of the platelet suspensions. Pure platelets are not able to induce a primary antibody response. The present study shows that the platelets, however, can be recognized by the immune system but they induce a suppression of the response. Mice pretreated with donor platelets will not give a primary antibody response upon a subsequent injection of donor leukocytes and the survival of donor skin grafts will be prolonged. Similar results were obtained by pretreatment of the responder mice with heat-treated donor leukocytes. Furthermore, repeated injections of heat-treated leukocytes of the recipient strain to the donor before bone marrow grafting, will delay graft-versus-host mortality. These data show that cells which have only class I antigens on their surface and no activating class II antigens, induce a suppression of the response against class I antigens.     

Diagnosis and treatment of immunological platelet refractoriness by histocompatibility

Immunological platelet refractoriness occurs when polytransfused patients develop antibodies against donors’ HLA class I antigens, HPA (human platelet antigens) and few cases against both systems. Flow cytometry crossmatch with the patient serum against platelets from several donors can determine whether the refractoriness is or is not of immunological origin. Patients with moderate sensitization will be given transfusions from donors with a negative platelets crossmatch; those who are hypersensitized will need to have antibodies assessed against a reactivity panel (RP) for HLA class I and HPA. The patient must be typed for HLA and HPA in order to identify best donors. We have compiled a list of 500 donors registered at our blood bank with known HLA and HPA profiles. Pre-transfusion crossmatch is performed against donors selected virtually, transfusing those who are negative.We analyzed 75 patients with refractoriness, 67% (50/75) of whom had anti-HLA or anti-HPA antibodies and 56% (28/50) were hypersensitized, with RP ≥ 80%.The diagnosis of the immunological refractoriness and the compatibility between donor and recipient allowed efficient transfusions for all patients.     

A simple method for inhibiting ABO antibodies in sera used for platelet crossmatching

Sometimes it is necessary to crossmatch and transfuse ABO-incompatible platelets As IgG anti-A and anti-B sometimes react with platelets from group A or B donors, these reactions can confuse the interpretation of crossmatching, which is designed to detect HLA or platelet-specific antibodies. Methods previously described to overcome this problem have been complex. Neutr-ABR, which contains A and B blood group substances from porcine and equine sources, can be used to neutralize anti-A and/or anti-B in sera used to crossmatch ABO-incompatible platelets. This simple technique has been used for many years in red blood cell (RBC) serology and involves simply adding 1 volume of Neutr-AB to 5 volumes of serum and incubating at room temperature for 5 minutes. In this study, 13 of 65 (20%) random group O donor sera reacted against random group A platelets and 6 of 67 (9%) random group O donor sera reacted against random group R platelets. All of the anti-A and anti-B reactions were inhibited by Neutr-AB when tested against group A or B platelets by solid-phase ELISA. The amount of A antigen on platelets and the levels of anti-A in group B or O sera can vary considerably, so we investigated the extent of the problem in platelet crossmatching. Sixty percent of 20 random group O sera reacted with platelets from a donor, who was selected because of the strong A antigen present on the platelets. Eighty-six percent of randomly selected group A platelets reacted with a group O serum containing strong anti-A. Neutr-AB was found not to inhibit 1 anti-P1A1 and 6 anti-HLA.     

The effect of pretransplant platelet transfusions on renal allograft survival and sensitization in dogs

Mongrel dogs were given either whole blood (50 ml) or platelets prepared from 50 ml blood intravenously on three occasions before transplantation of a kidney from a different donor to that of the blood or platelets. All dogs were given azathioprine and prednisolone after transplantation. 60 X 10(8) platelets were obtained from 50 ml blood and the leucocyte contamination was less than one leucocyte per 10(5) platelets. The mean survival of kidney allografts in non-transfused dogs was 11.6 days (10 dogs), in dogs pretreated with whole blood 26.6 days (5 dogs), and in dogs pretreated with platelets 29.2 days (5 dogs). Sensitisation occurred in 3 of 5 dogs given whole blood and in 2 of 5 dogs given platelets. Thus pretreatment with a relatively pure preparation of platelets will produce prolongation of survival of third party renal allografts in mongrel dogs given azathioprine and prednisolone, comparable to that produced by whole blood. However, sensitisation was produced by the platelet preparation, presumably due to the minimal leucocyte contamination, which might also be responsible for the suppressive effect of this platelet pretreatment protocol.     

Decrease in platelet serotonin level in uterine cervix cancer patients

Platelet serotonin (5-HT) concentration was measured by HPLC with electrochemical detection in 46 women suffering from cancer of the uterine cervix and 16 matched controls. About 53% reduction (p < 0.05) was recorded in platelet 5-HT level in cancer patients against a control value of 1.29 +/- 0.16 (mean +/- S.E.) nmol per 10(9) platelets. Depletion of intraplatelet 5-HT was positively correlated with clinical stage of the disease although a modest rise (p > 0.05) in platelet 5-HT was observed in patients at stage I. Serotonin release from platelets following activation with thrombin was considerably increased in cancer patients (38.2% compared to 17.4% in controls). The results demonstrate progressive depletion of intraplatelet 5-HT in cervical cancer patients. In addition, their platelets release more 5-HT than the controls upon activation by thrombin.     

Secretion of the terminal complement proteins, C5-C9, by human platelets

The terminal complement components, C8 and C9, and to a lesser extent C5, C6, and C7, but minimal amounts of C3, were shown to be associated with washed human platelets. In unactivated platelets, the complement components were detected in the platelet pellet by hemolytic assays after centrifugation and disruption of the platelets by freeze-thawing. However, after platelets had been activated by collagen, thrombin, or aggregated IgG to induce aggregation, the complement components were released into the supernatant. The rank order of hemolytic activity of C9, C8, C7, C6, and C5 detected in the supernatants of activated platelets was quite different from that found in serum from the same donors, in the same assays. In particular, the serum C7 hemolytic titer was more than twice the serum C9 hemolytic titer, whereas the activity of C9 detected from platelets was more than twice that of C7. This argues against a purely nonspecific uptake of these proteins by platelets from plasma. The functional role of terminal complement components released from platelets during activation is unknown, but it is tempting to speculate that these proteins may have a role in platelet-dependent immunological tissue injury. Because the C5b-9 membrane attack complex activates platelets, it is possible that release of terminal complement proteins serves to amplify platelet activation and may also play a role in diseases in which complement membrane attack complexes have been implicated.     

The efflux of 86Rb and [3H]5-HT from human platelets during continuous perfusion: effects of potassium-induced membrane depolarization and thrombin stimulation

In order to study the ionic efflux or granule release from human platelets following pulse exposure to various stimuli, a method for continuous perfusion of platelets was developed. The method was applied to compare the effects of membrane depolarization and thrombin stimulation on the release of 86Rb and [3H]5-HT. Washed and preloaded human platelets were placed on a membrane filter in a temperature controlled polypropylene chamber, and subsequently perfused with buffer. After an initial washout period the efflux of 86Rb or [3H]5-HT reached steady, low levels. K+ induced concentration dependent increases in 86Rb efflux, corresponding to a depolarization of the membrane potential, whereas the efflux of [3H]5-HT was unaltered. Thrombin induced concentration dependent increases in the efflux of both 86Rb and [3H]5-HT. Pretreatment with K+ 12 or 30 mM did not alter the [3H]5-HT efflux induced by thrombin 0.1 U ml-1. Scanning electron micrographs of platelets on the filter showed that the unstimulated platelets had regular shape, whereas after addition of thrombin there was formation of pseudopods and minor aggregates. The effect of potassium-induced membrane depolarization on platelet aggregation was also studied. High concentration of K+ did not induce aggregation or shape change during 2 or 10 minutes of incubation. K+ had little or no effect on aggregation induced by ADP 2 microM or thrombin 0.4 U ml-1. The results from release experiments and aggregation tests argue against an immediate coupling between membrane potential and platelet reactivity.     

Reactivity of mouse antibodies against bromelain-treated mouse erythrocytes with thrombin-treated mouse platelets.

The reactivity of mouse antibodies against bromelain-treated mouse erythrocytes (BrMRBC) with mouse platelets before and after thrombin treatment was assessed by flow cytometry. Anti-BrMRBC antibodies could bind to thrombin-treated platelets, although normal platelets were also weakly reactive with the antibodies. The binding of anti-BrMRBC antibodies to platelets was confirmed by complement-dependent lysis. It is suggested that thrombin-activated platelets may be a real target for anti-BrMRBC antibodies.     

Cross-Reactive HL-A Antibodies. Separation of Multiple HL-A Antibody Specificities by Platelet Adsorption and Acid Elution

A method of isolating and concentrating monospecific antibody from multispecific HL–A antiserum by adsorption with and elution from platelets is described. One unit of platelets containing approximately 1 × 10¹¹ platelets is sufficient to absorb one unit (300 ml) of serum of low titer. Antibodies directed against antigens determined at separate loci are readily separated by adsorption with appropriately selected platelets, whereas antibody specificities directed against antigens determined by the same locus are usually not separable. Successful adsorption and elution of an antibody present in duospecific antisera depends not only upon the presence of a particular HL–A antigen on the platelets used for adsorption but also upon the absence of any antigen group which cross-reacts with the second antibody specificity.     

Influence of platelets and platelet microbicidal protein susceptibility on the fate of Staphylococcus aureus in an in vitro model of infective endocarditis

Several lines of evidence indicate that platelets protect against endovascular infections such as infective endocarditis (IE). It is highly likely that a principal mechanism of this platelet host defense role is the release of platelet microbicidal proteins (PMPs) in response to agonists generated at sites of endovascular infection. We studied the ability of platelets to limit the colonization and proliferation of Staphylococcus aureus in an in vitro model of IE. Three isogenic S. aureus strains, differing in their in vitro susceptibility to thrombin-induced platelet microbicidal protein-1 (tPMP), were used: ISP479C (parental strain; highly susceptible to tPMP [tPMP(s)]); ISP479R (transposon mutant derived from ISP479; tPMP resistant [tPMP(r)]); or 757-5 (tPMP(r) transductant of the ISP479R genotype in the ISP479 parental background). Time-kill assays and in vitro IE models were used to examine the temporal relationship between thrombin-induced platelet activation and S. aureus killing. In time-kill studies, early platelet activation (30 min prior to bacterial exposure) correlated with a significant bactericidal effect against tPMP(s) ISP479C (r(2) > 0.90, P < 0.02) but not against tPMP(r) strains, ISP479R or 757-5. In the IE model, thrombin activation significantly inhibited proliferation of ISP479C within simulated vegetations compared to strains ISP479R or 757-5 (P < 0.05). The latter differences were observed despite there being no detectable differences among the three S. aureus strains in initial colonization of simulated vegetations. Collectively, these data indicate that platelets limit intravegetation proliferation of tPMP(s) but not tPMP(r) S. aureus. These findings underscore the likelihood that platelets play an important antimicrobial host defense role in preventing and/or limiting endovascular infections due to tPMP(s) pathogens.     

Interferon-�� increases monocyte migration via platelet�Cmonocyte interaction in murine intestinal microvessels

The aim of this study was to investigate the effect of interferon (IFN)‐α on recruitment of platelets and monocytes within the murine small intestinal venular endothelium. Monocytes were isolated from bone marrow of C57B6 mice. Platelets were collected from murine blood. Rolling and adhesion to submucosal microvessels in the small intestine were examined under an intravital fluorescence microscope after injection of fluorescein‐labelled monocytes or platelets. In some mice, IFN‐α (5 × 10⁵U/kg) was administered intraperitoneally. After treatment with an antibody against P‐selectin, changes in monocyte and platelet migration were also investigated. Changes in monocyte migration under the condition of thrombocytopenia were also investigated. Platelets and monocytes interacted with murine intestinal microvessels, although only few platelets and monocytes showed migration behaviour. Intraperitoneal injection of IFN‐α enhanced the migration of both platelets and monocytes in the intestinal microvessels. Pretreatment with anti‐P‐selectin attenuated the increase in migration of platelets and monocytes induced by administration of IFN‐α. Thrombocytopenia decreased the rolling ratio of monocytes, suggesting that the effect of IFN‐α on migration was P‐selectin‐dependent, derived from both the endothelium of microvessels and platelets. The results of this study suggest that IFN‐α acts as a potent proinflammatory agent via its stimulatory effect on the endothelium–platelet–monocyte interaction in intestinal microvessels by a P‐selectin‐dependent mechanism.     

A monoclonal antibody-based enzyme immunoassay for human GMP-140/P-selectin.

Two hybridoma cell lines producing monoclonal antibodies WGA-1 and PL7-6, reactive only with thrombin-stimulated human platelet have been established. Both these antibodies were investigated for their specific reactivity against GMP-140, based on the amino acid composition analysis of immunopurified antigen and N terminal amino acid sequencing of its protease fragments. A two-site enzyme immunoassay for quantification of human GMP-140 was developed using WGA-1 monoclonal antibody immobilized on 96-well microplates and horseradish peroxidase-labeled PL7-6 monoclonal antibody as detector. The assay was able to measure GMP-140 in serum and plasma with a sensitivity of about 5 ng/ml and a precision better than 10%. This assay will be useful for the detection of GMP-140 derived from platelets or endothelium in biological fluids and tissue extracts.     

On the mechanism of thrombocytopenic purpura in sexually active homosexual men

Thrombocytopenic purpura has recently been noted in sexually active homosexual men. To elucidate the pathogenesis of thrombocytopenic purpura in this population, we compared the disorder in 33 homosexual men with that in 23 patients (15 women and 8 men) thought to have classic autoimmune thrombocytopenic purpura. The homosexual group had 3.8-fold higher levels of platelet-bound IgG and 4.2-fold higher levels of platelet-bound complement than the patients with autoimmune thrombocytopenic purpura. Eluates from the platelets of only 1 of 10 homosexual patients reacted in vitro with normal platelets, as compared with those from the platelets of 12 of 15 patients with autoimmune thrombocytopenic purpura. Twenty-one of 24 homosexual patients (88 per cent) had elevated serum levels of immune complexes that were capable of binding to platelets, whereas none of 5 patients with autoimmune thrombocytopenic purpura had circulating immune complexes. The IgG fraction of positive serum samples from three homosexual patients did not bind to normal platelets, whereas that from the positive serum of two patients with autoimmune thrombocytopenic purpura and one woman in whom isoimmune antiplatelet antibody developed during pregnancy (studied as a positive control) did bind to normal platelets. We conclude that, whereas classic autoimmune thrombocytopenic purpura involves antiplatelet IgG directed against platelet antigenic determinants, the thrombocytopenic purpura that occurs in sexually active homosexual men is usually caused not by antiplatelet IgG but probably by the nonspecific deposition of complement and immune complexes on platelets.     

Purification and in vitro activities of rabbit platelet microbicidal proteins.

Recent in vitro studies have demonstrated that rabbit platelets release a small, cationic antimicrobial protein in response to thrombin stimulation under physiological conditions (M. R. Yeaman, S. M. Puentes, D. C. Norman, and A. S. Bayer, Infect. Immun. 60:1202-1209, 1992). This observation prompted our present investigation, focused on determining the array of antimicrobial proteins contained within rabbit platelets and their in vitro activity against common bloodstream pathogens. A group of small (6.0- to 9.0-kDa), cationic proteins with in vitro antimicrobial activity was purified from whole and thrombin-stimulated rabbit platelets by gel filtration and reversed-phase high-performance liquid chromatography. Purified proteins in micromolar concentrations (10 to 40 microg/ml) exerted in vitro microbiostatic and/or microbicidal activities against Staphylococcus aureus, Escherichia coli, and Candida albicans in a dose-dependent manner. The antimicrobial activities of proteins purified from rabbit platelet acid extracts were generally inversely related to pH, with maximal activity observed at pH 5.5. In contrast, the predominant protein isolated from thrombin-stimulated rabbit platelets, though biochemically and microbiologically similar to proteins extracted by acid, exhibited antimicrobial activities which were modestly enhanced at pH 7.2 compared with pH 5.5. Amino acid compositional analyses in combination with molecular mass determinations suggest that the majority of these proteins are distinct molecules not derived from a single common precursor. Collectively, these data indicate that rabbit platelets contain proteins which exert potent in vitro antimicrobial activity against bacterial and fungal pathogens which commonly invade the bloodstream. Moreover, several of these proteins were released from platelets stimulated with thrombin under physiological conditions and exerted potent antimicrobial activities in physiological pH ranges. These observations support the hypothesis that platelets serve an important role in host defense against infection, via localized release of antimicrobial proteins in response to stimuli associated with tissue injury or microbial colonization.     

Competitive inhibition of T-cell mediated lympholysis by platelets

Platelets are known to possess serologically detectable HLA-A, B, C (class I) antigens, but not HLA-DR (class II) antigens. We have used platelets as non-labelled (cold) competitors for cell-mediated lympholysis directed against determinants on PHA (3 days) and PWM (7 days) ⁵¹Cr-labelled (hot) lymphoblasts, i.e. T and B lymphoblasts, respectively.
It is found that platelets are able to incompletely inhibit cytolysis against T-lymphoblasts, but not B-lymphoblasts. The inhibition is immunologically specific in the sense that only platelets autologous to the original stimulator cell block, while platelets autologous to the original responder cell do not. The immunogenetic specificity of platelet blocking is unknown at present, since no allogeneic third party platelets have been investigated. It is further found that platelets do not inhibit cytolysis by cytotoxic lymphocytes previously qualified to identify HLA-non A, B, C, D/DR determinants on T-lymphoblasts.
Like in serology, platelets have to mature (≥ 7 days) in order to obtain optimal results. Since platelets are easy to procure and maintain their reactivity for months by simple storage in saline at 4°C, platelets may be used to screen for cell-mediated lympholysis against HLA-class 1, II or "new" determinants.     

Role of membrane glycoproteins in the interaction of blood platelets with the vessel wall--the study on platelet adhesion to in vitro cultured subendothelial matrix

Adhesion of platelets to the subendothelium is an essential step in hemostasis and thrombosis. Several receptors for adhesive macromolecules have been identified on platelets and are included in the integrin family. To clarify the role of platelet membrane glycoproteins in the interaction of platelets with the subendothelium, 51Cr-labeled platelet adhesion assay and antibody-blocking experiments were performed by using in vitro cultured subendothelium under the static condition. The platelet adhesion in this assay was inhibited by anti-GPIa (VLA-2), GPIc (VLA-5) and -GPIc'-(VLA-6) antibodies, while anti-GPIb and -GPIIb/IIIa antibodies had no effect. Platelets from the patients with Glanzmann's thrombasthenia could also attach to the subendothelium, whereas those from a patient whose platelets lacked GPIa failed to attach to the extracellular matrix (ECM). The monoclonal antibodies against fibronectin and laminin which recognized the cell binding domain of these molecules inhibited the platelet adhesion when they were pre-treated with ECM. Furthermore, antibody-blocking experiments revealed that the percent inhibition by the combination of anti-GPIa, -GPIc and -GPIc' antibodies used herein was approximately 75%. They did not completely inhibit the attachment. These results suggest that the interactions of collagen, fibronectin and laminin with their receptors on platelets are involved in the mechanism of platelet adhesion to subendothelium.     

Enzyme-linked immunosorbent assay of CS-518, a thromboxane synthetase inhibitor, in rabbit plasma and platelets.

A competitive enzyme-linked immunosorbent assay (ELISA) was developed for determination of CS-518, a novel thromboxane synthetase inhibitor. Antisera against CS-518 were obtained from rabbits immunized with bovine serum albumin linked to CS-518 via carboxylic acid introduced into the imidazolyl ring (for ELISA-1) or via 6-carboxylic acid directly (for ELISA-2). Each of two CS-518 derivatives was conjugated to horseradish peroxidase by a N-succinimidyl ester method, and it was used as a labeled-antigen in homogeneous combination with antisera. In ELISA-1, CS-518 was detectable in a range of 5pg-1ng, and all cross-reactivities with main metabolites were less than 5%, in contrast to high affinity to the taurine and glucuronic acid conjugates of CS-518 in ELISA-2. Validity of ELISA-1 was confirmed by a high-performance liquid chromatography and ELISA-1 enabled specific determination of CS-518 in plasma samples deproteinized by methanol. When ELISA-1 was applied to determine CS-518 in platelets after oral administration to rabbits, CS-518 uptake up to maximum capacity in platelets (4.2-5.4 x 10(6) M) and slow elimination of CS-518 from platelets (T1/2 = 36-41 hr) were observed independent of CS-518 doses. These results confirm that CS-518 binds to thromboxane synthetase in platelets with high affinity.     

Complement-dependent and cell-dependent antiplatelet humoral antibody in sera from multi-transfused patients.

Sera from eight multi-transfused patients, who were resistant to platelet trans-fusions from allogeneic donors, were tested for antiplatelet humoral antibody. Complement-dependent cytotoxic humoral antibody against platelets was found in sera from six of eight patients. Cell-dependent cytotoxic humoral antibody against platelets was found in sera from four of seven of these patients. Sera from two patients had cell-dependent antiplatelet activity but no detectable complement-dependent antiplatelet activity. Results of this study argue that both complement-dependent and cell-dependent humoral immunity may be important pathways for in vivo resistance to platelets.     

Volume regulatory mechanisms of human platelets

Platelets were found to defend themselves against hypoosmotic swelling by a regulatory volume decrease mechanism, similar to that of T-lymphocytes. An oligomycin C sensitive Cl- -channel and a Ca2+-dependent, quinine- and trifluoperazine-sensitive K+-channel play a significant role in the process: platelets lose K+ and Cl- (and consequently water) through these. Activation of platelets accelerates the volume regulatory mechanism in spite of the fact that the Na+/H+ exchange process, activated simultaneously, works against volume decrease. The most rapid volume regulation was observed in activated platelets with inhibited Na+/H+ exchange process.