Thomas J. Kunicki

A Study of Variables Affecting the Quality of Platelets Stored at “Room Temperature”

The effect of variables associated with the donor and with methods of collecting, processing, and storing platelets on the quality of platelets kept at ambient temperature was studied. Changes in structural integrity of platelets, decrease in pH, loss of aggregability, and kinetics in vivo of platelets tagged with 51Cr were used as indicators of the tolerance of platelets to storage. A platelet concentration of less than 2.5 × 106 per cu mm, a temperature of storage less than 24 C, and continuous, gentle, agitation were found to be essential for satisfactory preservation of platelet integrity, function, and posttransfusion survival. Platelets from female donors tolerated storage less well than did platelets from male donors, possibly because the lower hematocrit of blood collection from females resulted in greater initial acidity of the concentrate. A number of other variables analyzed appear to be of little or no consequence for successful platelet storage.

Studies of Platelet Concentrates Stored at 22 C and 4 C

As measured by stability of pH, aggregability, and structure, concentrated platelets are better preserved for 72 hours at 4 C than at 22 C. While survival in vivo, as determined with 51Cr, of platelets stored at room temperature is extremely variable, survival of platelets stored at 4 C is invariably shortened to two to three days. In the treatment of bleeding in thrombocytopenic patients, however, platelets kept at 4 C for 24 to 72 hours are superior to those kept at 22 C with respect to their capacity to shorten the bleeding time and effect hemostasis. The relative ineffectiveness of platelets kept at 22 C for 72 hours appears to be the result of a functional defect which develops during storage at ambient temperature.

Isolation and immunologic characterization of the human platelet alloantigen, P1A1.

Abstract Platelets from patients with Glanzmanns thrombasthenia, acongential disorder of platelet function, are deficient in the membrane alloantigen. P1a1 (Zwa). present on platelets of nearly all normal subjects. Because thrombasthenic platelets are also deficient in two membrane glycoproteins, designated IIb and IIIa. it has been suggested that one or both of these glycoproteins may carry the P1a1 antigenic determinant. To address this question, the P1a1 antigen was isolated by sequential lectin affinity chromatography of sodium deoxycholate extracts of platelet membranes, by indirect immunoprecipitation of Nonidet P40 extracts of lactoperoxidase-iodinated intact platelets, and by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane preparations. By each of the three separatory procedures, the P1a1 antigenic marker was shown to be associated with glycoprotein IIIa. Studies of the effects of proteolytic enzymes, disulfide-reactive agents and temperature on P1a1 activity and of the effect of various sugars on the P1a1-anti-P1a1 reaction indicate that the P1a1 antigen, situated on a portion of the molecule susceptible to cleavage in situ by trypsin, but not by chymotrypsin. bromelain, or papain, requires intact disulfide bonds for its full expression, and is probably determined by a polypeptide sequence in GPIIIa. Virtually all of the P1a1 activity of platelets appears to be located on the external plasma membrane. P1a1 appears to be the first alloantigen to be assigned to a specific platelet membrane constituent and the third peptide-determined alloantigen to be assigned to a human cell membrane protein.

Effects of monoclonal antibodies against the platelet glycoprotein IIb/IIIa complex on thrombosis and hemostasis in the baboon.

To assess the hemostatic consequences and antithrombotic effectiveness of blocking the platelet glycoprotein (GP) IIb/IIIa receptor for fibrinogen and other adhesive glycoproteins in vivo, well characterized murine monoclonal antibodies against the platelet GP IIb/IIIa complex, AP-2 and LJ-CP8, were infused intravenously into baboons. Four animals each received doses of 0.2, 0.4, and 1.0 mg/kg of purified AP-2 IgG, and three animals were given 1.0 mg/kg of the F(ab)2 fragment of AP-2. Five additional animals were given 10 mg/kg LJ-CP8 IgG. At the highest dose, radiolabeled AP-2 IgG bound to an average of 33,000 sites on the circulating platelets. Serial measurements included platelet count, bleeding time, platelet aggregation (induced by ADP, collagen, and gamma-thrombin), and 111In-platelet deposition onto Dacron vascular grafts. Bleeding times were markedly prolonged after injection of 1.0 mg/kg AP-2 IgG (19.2 +/- 3.4 min), 1.0 mg/kg AP-2 F(ab)2 (16.5 +/- 1.8 min), and 10 mg/kg LJ-CP8 (greater than 30 min) vs. control studies (4.6 +/- 0.2 min), and remained prolonged for 48 h. With each antibody platelet aggregation was initially reduced or absent, with partial recovery over 48 h in a manner that was inversely related to dose. AP-2, both whole IgG and F(ab)2 fragment, but not LJ-CP8, caused a dose-dependent reduction (20-46%) in the circulating platelet count over 24 h. Neither AP-2 nor LJ-CP8 caused a reduction in intraplatelet platelet factor 4, beta-thromboglobulin, or [14C]serotonin. Graft-associated platelet thrombus formation was reduced by 73% (1.0 mg/kg AP-2 IgG and 10 mg/kg LJ-CP8) and 53% (1.0 mg/kg AP-2 F(ab)2) relative to control values. In contrast, neither heparin (100 U/kg) nor aspirin (32.5 mg/kg twice a day) showed antithrombotic efficacy in this model. Thus, antibodies that functionally alter the platelet GP IIb/IIIa complex may produce immediate, potent, and transient, antihemostatic, and antithrombotic effects.

Diagnosis of Bernard-Soulier syndrome and Glanzmann's thrombasthenia with a monoclonal assay on whole blood.

Two hereditary platelet disorders, Bernard-Soulier syndrome and Glanzmanns thrombasthenia, are characterized by selective deficiencies of platelet membrane glycoproteins. Murine monoclonal antibodies were developed against platelet membrane glycoprotein Ib and against the glycoprotein IIb/IIIa complex. A rapid whole blood assay for the deficiency of these glycoproteins was developed and used to study whole blood samples from six patients with Glanzmanns thrombasthenia and three patients with Bernard-Soulier syndrome. Patients with type I and type II Glanzmanns thrombasthenia were easily detectable with this assay. This permits the diagnosis of these disorders on 200 microliters of whole blood within 2 h of blood sampling.

On the association of the platelet-specific alloantigen, Pena, with glycoprotein IIIa. Evidence for heterogeneity of glycoprotein IIIa.

Neonatal alloimmune thrombocytopenic purpura associated with a new platelet-specific alloantigen Pena has been reported. We now provide direct evidence that the Pena determinant is associated with glycoprotein (GP) IIIa, but that it is distinct from epitopes that define the PlA system. By ELISA wherein monoclonal antibodies specific for GPIIb (Tab) and specific for GPIIIa (AP3) were used to capture and hold antigens from a platelet lysate prepared under conditions that generate free GPIIb and GPIIIa, anti-Pena reacted with GPIIIa held by AP3 but not with GPIIb held by Tab. In an alternative ELISA where purified GPIIIa from both PlA1-positive and PlA1-negative platelets were used individually as antigen, anti-Pena reacted with both allelic forms of GPIIIa. By radioimmuno-precipitation, anti-Pena precipitated a single surface-labeled membrane protein with electrophoretic characteristics in sodium dodecyl sulfate-polyacrylamide gels, under nonreduced or reduced conditions, identical to those of GPIIIa. By fluorocytometry, platelets from several donors, regardless of PlA phenotype, bound an amount of anti-Pena roughly equivalent to one-half that amount of anti-PlA1 bound by PlA1 homozygous (A1/A1) platelets and roughly equal to that amount of anti-PlA1 bound by PlA1 heterozygous (A1/A2) platelets. Using platelets from donors typed homozygous for PlA1 and Pena in a quantitative indirect binding assay, 14-24,000 molecules of anti-Pena and 41-51,000 molecules of anti-PlA1 were bound per platelet at saturation. Anti-Pena completely inhibited ADP-induced aggregation of Pena-positive platelets, regardless of PlA phenotype. These results indicate that the Pena determinant is associated with GPIIIa but distinct from PlA.

Independent modulation of von Willebrand factor and fibrinogen binding to the platelet membrane glycoprotein IIb/IIIa complex as demonstrated by monoclonal antibody.

In this study we have used two new monoclonal antibodies, designated LJP5 and LJP9, as well as a previously described one, AP2, all specific for the platelet membrane glycoprotein (GP)IIb/IIIa complex. None of them reacted with dissociated GPIIb or GPIIIa. The monovalent Fab fragment of both LJP5 and LJP9 bound to unstimulated platelets in a saturable manner, but binding was markedly decreased after platelets had been incubated at 37 degrees C in the absence of added extracellular calcium. The binding of LJP9 was not affected by AP2, but was blocked by excess LJP5. On the contrary, the binding of LJP5 was blocked in the presence of both AP2 and LJP9. Thus, these antibodies bound to distinct epitopes of GPIIb/IIIa. At saturation, the binding to unstimulated platelets was between 2.41 and 10.9 X 10(4) molecules/platelet for LJP5 and between 3.47 and 9.1 X 10(4) molecules/platelet for LJP9 (range of 11 and 10 experiments, respectively). Binding increased up to 50% after thrombin stimulation. The estimated association constant, Ka, was 2.7 X 10(7) M-1 for LJP5 and 3.85 X 10(7) M-1 for LJP9. Both LJP5 and LJP9 partially inhibited the association of 45Ca2+ with the surface of unstimulated platelets. Moreover, both antibodies blocked the binding of von Willebrand factor (vWF) to stimulated platelets, whereas only LJP9, but not LJP5, blocked fibrinogen binding. LJP9 was also a potent inhibitor of platelet aggregation, whereas LJP5 was without effect in this regard. The results of the present study demonstrate that independent modulation of vWF and fibrinogen binding to stimulated platelets can be attained with monoclonal antibodies directed against distinct epitopes of GPIIb/IIIa.

Activation of the Integrin Involves a Discrete Cation-binding Site That Regulates Conformation

“Activation” of integrins is involved in the dramatic transition of leukocytes and platelets from suspension to adhesion. The integrin αβ is not known to take part in this sort of transition, even though it shares its β subunit with αβ, the activable integrin on platelets. In the context of a constitutively adhered cell, changes in activation state may be more subtle in their effects, but nonetheless important in regulating cell behavior. We hypothesized that αβ can undergo conformational changes analogous to those associated with αβ activation. Accordingly, we examined αβ on the surface of M21 cells (a human melanoma cell line) and found that, like αβ, it can undergo conformational changes upon binding of a ligand analog and can be activated for ligand binding and migration by a monoclonal antibody directed against β. Modulation of the binding of this activating antibody, AP5, ligand binding, and antibody-mediated activation all are associated with a discrete cation-binding site shared in both αβ and αβ. Based on a measured K, this site has an apparent K for calcium of approximately 20 μM. At physiological levels of calcium, about 40% of the total αβ on a cells surface is in a conformation detected by AP5. The data suggest a model for both αβ and αβ function in which the molecule can exist in either of (at least) two conformational states, one stabilized either by AP5 or ligand binding, refractory to calcium binding, and enhanced for ligand recognition, the other stabilized by calcium binding and refractory to AP5 and ligand binding. Functional analysis suggests that AP5 activates αβ by preventing occupation of this calcium site, and that the activated form of αβ differs functionally from the basal form. The active form is more conducive to migration and the basal to tight adhesion.

Absence of the platelet receptor for drug-dependent antibodies in the Bernard-Soulier syndrome.

The platelet membrane receptor for quinidine- and quinine-dependent antibodies was studied in three patients with the Bernard-Soulier syndrome (BSS) and in normal subjects with immunologic techniques based on the release of 51Cr from labeled platelets. The receptor could not be detected on BSS platelets but was present on platelets from each of 180 normal subjects. BSS platelets reacted normally with other allo- and autoantibodies. In confirmation of previous reports, BSS platelets were found to be deficient in glycoproteins Ib and Is. However, after apparently total cleavage of these proteins from the membrane of normal platelets by controlled hydrolysis with trypsin or chymotrypsin, 80% of the drug-dependent antibody receptor activity was retained. These observations suggest the existence of an additional, hitherto unrecognized membrane defect in Bernard-Soulier platelets.

Deletion of the Platelet-Specific Alloantigen PlA1 from Platelets in Glanzmann's Thrombasthenia

Expression of a Platelet-specific alloantigen (Pl(A1)) was studied in five unrelated patients with Glanzmanns thrombasthenia using immunologic techniques based on release of (51)Cr from tagged platelets by Pl(A1)-specific antibody. Less than 1% of the normal quantity of Pl(A1) could be detected on platelets of patients 1, 2, and 3; platelets from patients 4 and 5 contained 22 and 12% of normal levels, respectively. After treatment with bromelain, platelets from patients 4 and 5, but not those from patients 1, 2, and 3, released (51)Cr as well as normal Pl(A1)-positive platelets when exposed to anti-Pl(A1). Platelets from each of the five patients reacted normally with drug-dependent antibodies and with autoantibodies specific for platelets. Polyacrylamide gel electrophoresis of thrombasthenic platelets showed marked deficiencies of glycoproteins IIbalpha and III (P < 0.0005), confirming recent reports of others. Deficiency of the two proteins as determined by gel scanning was more pronounced in patients 1, 2, and 3 than in patients 4 and 5. Normal levels of glycoproteins IIbalpha and III were found in platelets from normal subjects negative for Pl(A1). These observations are consistent with the possibility that the Pl(A1) antigen is located on one or both of the glycoproteins lacking in Glanzmanns thrombasthenia, although other explanations are possible. They further suggest that patients with thrombasthenia may be heterogeneous in respect to the degree to which these glycoproteins are deleted. The Pl(A1) antigen can be measured with considerable precision and may provide a marker useful for the diagnosis and study of Glanzmanns disease.