A platelet alpha-granule membrane protein (GMP-140) is expressed on the plasma membrane after activation

We have previously characterized a monoclonal antibody, S12, that binds only to activated platelets (McEver, R.P., and M.N. Martin, 1984, J. Biol. Chem., 259:9799-9804). It identifies a platelet membrane protein of Mr 140,000, which we have designated as GMP-140. Using immunocytochemical techniques we have now localized this protein in unstimulated and thrombin-stimulated platelets. Polyclonal antibodies to purified GMP-140 were used to enhance the sensitivity of detection. Nonpermeabilized, unstimulated platelets, incubated with anti-GMP-140 antibodies, and then with IgG-gold probes, showed very little label for GMP-140 along their plasma membranes. In contrast, thrombin-stimulated platelets exhibited at least a 50-fold increase in the amount of label along the plasma membrane. On frozen thin sections of unstimulated platelets we observed immunogold label along the alpha-granule membranes. We also employed the more sensitive technique of permeabilizing with saponin unstimulated platelets in suspension, and then incubating the cells with polyclonal anti-GMP-140 antibodies and Fab-peroxidase conjugate. Alpha-granule membranes showed heavy reaction product, but no other intracellular organelles were specifically labeled. These results demonstrate that GMP-140 is an alpha-granule membrane protein that is expressed on the platelet plasma membrane during degranulation.     

Flow cytometric analysis of circadian changes in platelet activation using anti-GMP-140 monoclonal antibody.

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-alpha-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Flow Cytometric Analysis of Circadian Changes in Platelet Activation Using Anti-Gmp-140 Monoclonal Antibody

The hemostatic activity of blood shows a circadian variation with a higher frequency of acute coronary events in the morning. The thrombotic tendency of blood is influenced by many factors, including platelets. Diurnal changes of in vivo platelet activation were investigated by whole blood flow cytometry in 10 young healthy male volunteers using anti-GMP-140 (anti-α-granule membrane protein 140 kD) monoclonal antibody at 3h intervals from 06:00 to 24:00. We also studied circulating platelet aggregates to investigate whether there exists a similarity between the results of these methods. Results of flow cytometric analysis indicate that there is an increase in platelet activation during the period from 06:00 to 09:00. Platelet activation then decreases gradually during the period from noon to midnight. These changes are accompanied by a similar trend in circulating platelet aggregates. This suggests that GMP-140 expression on platelets is synchronized with or followed by platelet aggregate formation in vivo, and increased platelet activation may predispose individuals to thrombosis at this time.     

Flow cytometric studies of platelet responses to shear stress in whole blood

The objective of this work was to evaluate quantitatively the effects of flow on platelet reactions using a flow cytometric technique. Whole blood was exposed to well defined, laminar shear stress in a cone-and-plate viscometer in the absence of added agonists. Blood specimens were fixed with formaldehyde and incubated with two monoclonal antibodies. Antibody 6D1, specific for platelet membrane glycoprotein Ib (GPIb), was used to identify and enumerate platelets and platelet aggregates on the basis of their characteristic forward scatter and 6D1-FITC fluorescence profiles. Anti-CD62 antibody, specific for the granule membrane protein-140 (GMP-140), was used to measure platelet activation. Results showed platelet aggregation increasing with increasing shear stress with marked increase in this response for a pathophysiological stress level of 140 dyn/cm² and higher. This stress level also was the apparent threshold for formation of large platelet aggregates (“large” refers to particles larger than 10 μm in equivalent sphere diameter). These platelet responses to shear stress were insensitive to aspirin, but strongly inhibited by agents that elevate platelet cyclic adenosine monophosphate (cAMP) levels. Moreover, pre-incubation of whole blood with monoclonal antibodies that inhibit von Willebrand factor binding to GPIb or von Willebrand factor and fibrinogen binding to GPIIb/IIIa inhibited platelet aggregation. Aggregation induced by shear at 37° C was less in extent than at 23° C. At physiological shear stresses, whole blood was more susceptible to shear-induced platelet aggregation than platelet-rich plasma. This study reaffirms that flow cytometric methods have several important advantages in studies of shear effects on platelets, and extends the methodology to whole blood unaltered by cell separation methods.     

Correlated measurement of platelet release and aggregation in whole blood

We have used a technique for the simultaneous measurement of platelet activation and aggregation in whole blood using two-color immunofluorescence and flow cytometry to study the relationship between the release reaction and aggregation. A monoclonal antibody specific for the alpha granule membrane protein GMP-140 was used to measure the release reaction, and a monoclonal antibody specific for platelet membrane glycoprotein Ib (GPIb) was used to identify platelets and platelet aggregates. Aggregates were identified as particles expressing both levels of GPIb and size larger than that of resting single platelets. Anticoagulated whole blood was incubated with platelet agonists. At various times samples of the blood were removed and immediately fixed with paraformaldehyde. Blood that had been anticoagulated with ethylenediamine tetraacetic acid showed progressive release of platelets but little or no aggregation. However, blood anticoagulated with citrate or heparin showed correlated release and aggregation. The degree of aggregation was greater in heparin than in citrate. The expression of GPIb and GMP-140 increased in direct proportion to the size of the aggregates. Aggregates were observed varying in apparent diameter up to approximately 20 microns. During prolonged incubation there was progressive disaggregation of adenosine diphosphate (ADP)-induced aggregates. After disaggregation the proportion of GMP-140 negative single platelets increased, indicating that both released and nonreleased platelets participated in the aggregation. There was little or no disaggregation of phorbol myristate acetate (PMA)-induced aggregates. The relatively small size and reversibility of platelet aggregates that we have observed in whole blood may be relevant to phenomena occurring in vivo and in extracorporeal circulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of platelet concentrate during preparation and storage.

This review will discuss how stored platelets become activated and will examine their ability to function and survive in vivo, posttransfusion. Experimental methods which have been shown to alter platelets during storage will be detailed. Using beta-thromboglobulin (beta-TG) and surface adhesion receptors as markers, investigators have examined the activation changes in platelet concentrates during preparation and storage. Resuspension of the platelet pellet after isolation of platelet-rich plasma appears to play a major role in producing platelet activation and beta-TG release during preparation. However, there is a significant amount of interdonor variability in platelet activation even at this early stage of storage. Over 5 days of storage, platelets release approximately 50% of their beta-TG contents. Furthermore, between 40% and 60% of the platelets express the alpha-granule membrane protein, P-selectin (GMP-140), during storage, which is also indicative of platelet activation. These activation changes correlate to some degree with platelet recovery posttransfusion but clearly do not explain the full lesion of platelet storage. The surface density of two platelet membrane receptors, glycoproteins (GP) Ib and IIb/IIIa, also change with activation, although in opposite directions. Platelet surface GPIb decreases initially with storage and then recovers, perhaps due to its relocation to the platelet surface from an intracellular pool. In contrast to GPIb, mean platelet surface GPIIb/IIIa increases slightly during storage, probably as a consequence of platelet activation and release of alpha-granule GPIIb/IIIa to the surface. Some hypotheses are offered regarding how these activated platelets can continue to circulate after transfusion. Further exploration of the platelet storage lesion will hopefully provide needed answers and thus permit better treatment of hemostatic disorders in the future.     

GMP-140 binding to neutrophils is inhibited by sulfated glycans.

GMP-140 is a 140-kDa granule membrane glycoprotein localized to the alpha-granules of platelets and the Weibel-Palade bodies of endothelial cells. Expression of GMP-140 on the activated cell surface has been shown to mediate the adhesion of thrombin-activated platelets to neutrophils and monocytes and the transient adhesion of neutrophils to endothelium. In contrast, fluid-phase GMP-140 strongly inhibits the CD18-dependent adhesion of tumor necrosis factor alpha-activated neutrophils to endothelium suggesting that GMP-140 can also serve an anti-adhesive function. In the present report, it is demonstrated that fluid-phase GMP-140 which exists predominantly as a tetramer binds to a single class of high affinity receptor on neutrophils and HL60 cells. Binding of 125I-labeled GMP-140 to neutrophils and HL60 cells and the rosetting of neutrophils and HL60 cells by thrombin-activated platelets were inhibited by EDTA, excess unlabeled fluid-phase GMP-140, Fab fragments of an affinity-purified rabbit anti-GMP-140 antibody, and by the murine anti-GMP-140 monoclonal antibody, AK 4. Both neutrophil and HL60 GMP-140 binding and platelet rosetting were strongly inhibited by heparin, fucoidin, and dextran sulfate 500,000, were partially inhibited by dextran sulfate 5,000 and lambda- and kappa-carrageenan, but were not inhibited by chondroitins 4- and 6-sulfate. Since this sulfated glycan specificity is identical to that previously reported by us for GMP-140, the present results suggest that the sulfated glycan binding site and the neutrophil receptor binding site on GMP-140 are either identical or proximal.     

Structural and biosynthetic studies of the granule membrane protein, GMP-140, from human platelets and endothelial cells

GMP-140 is an integral membrane glycoprotein of apparent Mr = 140,000 located in secretory storage granules of platelets and vascular endothelial cells. When these cells are activated, GMP-140 redistributes from the membrane of the granules to the plasma membrane. To gain insight into the potential function of GMP-140, we examined aspects of its structure and biosynthesis. The amino acid composition of platelet GMP-140 revealed elevated numbers of cystinyl (6.1%), prolinyl (7.2%), and tryptophanyl (2.1%) residues. GMP-140 contained 28.8% carbohydrate by weight, distributed among N-acetylneuraminic acid, neutral sugar, and N-acetylglucosamine residues. Enzymatic removal of N-linked oligosaccarides reduced the protein's apparent Mr by more than 50,000. The biosynthesis of GMP-140 in HEL cells, which share biochemical features with megakaryocytes, was studied by pulse-chase labeling with [35S]cysteine followed by immunoprecipitation. HEL cells synthesized a heterogeneous GMP-140 precursor of 98-125 kDa which converted to a mature 140-kDa form within 40-60 min. Removal of high mannose oligosaccarides by endo-beta-N-acetylglucosaminidase H treatment reduced the apparent Mr of the precursor but not the mature protein. Tunicamycin-treated HEL cells synthesized three to four precursors of 80-92 kDa, suggesting the possibility of heterogeneity of GMP-140 at the protein level. Exposure of activated platelets to proteases followed by Western blotting indicated that most of the mass of GMP-140 was located on the extracytoplasmic side of the membrane. Our studies indicate that GMP-140 is a cysteine-rich, heavily glycosylated protein with a large extracytoplasmic domain. These features are compatible with a receptor function for the molecule when it is exposed on the surface of activated platelets and endothelial cells.     

Biochemical and immunohistochemical characteristics of CD62 and CD63 monoclonal antibodies. Expression of GMP-140 and LIMP-CD63 (CD63 antigen) in human lymphoid tissues.

During platelet secretion granule membrane glycoproteins are translocated to the plasma membrane. We report here the biochemical and immunohistochemical characterization of a panel of platelet-secretion-specific, CD62 and CD63 monoclonal antibodies (MoAb), which we raised to thrombin-activated platelets. The CD62 MoAb identify the alpha-granule membrane protein GMP-140, also designated platelet activation-dependent granule external membrane protein (PADGEM). The number of epitopes on thrombin-activated platelets ranged from 15,000 to 20,000. The CD63 MoAb recognize a 30-60 kDalton integral membrane protein of lysosomes. Due to its distinct localization, we have designated the CD63 antigen lysosome integral membrane protein, CD63 (LIMP-CD63). The number of epitopes on thrombin-activated platelets ranged from 9000 to 11,000. Expression of GMP-140, a member of the Selectin family (also referred as the LEC-CAM family) of adhesion molecules, and LIMP-CD63 was examined on human spleen, thymus and lymph node by immunohistochemistry. Both GMP-140 and LIMP-CD63 showed a wide distribution in lymphoid tissues; vascular endothelial cells and tissue compartments that were readily accessible to blood-borne components were uniformly positive for GMP-140 and LIMP-CD63. Furthermore, LIMP-CD63 was expressed in polymorphonuclear granulocytes and macrophages.     

Downregulation of GMP-140 (CD62 or PADGEM) expression on platelets by N,N-dimethyl and N,N,N-trimethyl derivatives of sphingosine.

GMP-140 (CD62 or PADGEM), a member of the selectin family, is a membrane glycoprotein in secretory granules of platelets and endothelial cells. When these cells are activated by agonists such as thrombin or AMP, GMP-140 is rapidly redistributed to the cell surface. The carbohydrate epitope defined by GMP-140 was identified as sialosyl-Le(x) (as for ELAM-1), which may play an essential role in adhesion of leukocytes or tumor cells on endothelial cells, through aggregation with platelets. Redistribution of GMP-140 from alpha-granules of platelets to the cell surface, induced by thrombin and PMA, was strongly inhibited by preincubation of platelets with N,N-dimethylsphingosine (DMS) or N,N,N-trimethylsphingosine (TMS) at 10-20 microM concentration for a brief period (5 min). Inhibition of GMP-140 redistribution to the cell surface by DMS or TMS was also detected by a cell adhesion assay using HL60 cells, which highly express sialosyl-Le(x); i.e., HL60 cells adhered on platelets activated by thrombin or PMA but not on platelets which were briefly preincubated with DMS or TMS followed by activation. The inhibitory effect of DMS or TMS on GMP-140 redistribution is not due to cytotoxicity, since the TMS-treated platelets were fully capable of aggregating in the presence of ristocetin. Sphingosine (SPN) and protein kinase C inhibitors such as H-7 and calphostin C showed weaker inhibitory activity than DMS and TMS. Our results indicate that both DMS and TMS could be useful reagents to inhibit cell surface expression of crucial selectins which promote adhesion of Le(x-) or sialosyl-Le(x)-expressing cells with platelets and endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of platelet activation using activation specific monoclonal antibodies

Platelets may become activated in a number of clinical disorders and participate in thrombus formation. Blood tests reflecting in vivo activation are therefore potentially useful in evaluating patients with thrombotic diseases. Three types of monoclonal antibodies have been described that react preferentially with activated platelets. Antibodies against a 53-kD lysosomal granule protein, and antibodies that recognize a 140-kD alpha-granule protein, are two types expressed on the platelet surface during secretion. A third type is not dependent on secretion and recognizes activation-dependent changes in the configuration or microenvironment of the platelet glycoprotein IIb/IIIa complex. Several procedures were used to detect platelet activation, using radiolabeled or fluorescent antibodies. In a number of disorders, changes in platelets, reflecting activation, could be detected. For the study of in vitro and in vivo platelet activation, these tests may be useful, but further studies are needed to confirm the power and efficiency of this approach compared to other routine tests.     

Topographic distribution of a granule membrane protein (GMP-140) that is expressed on the platelet surface after activation: an immunogold-surface replica study

Monoclonal and polyclonal antibodies have been developed that recognize a 140 kD glycoprotein on the plasma membrane of activated, but not unstimulated, platelets. This glycoprotein is found in resting platelets as an alpha-granule membrane protein and has therefore been named GMP-140. After thrombin stimulation, alpha-granules fuse with the surface-connected canalicular system and GMP-140 is redistributed to the plasma membrane. In the present study, we immunolabeled unstimulated and activated human platelets and analyzed the distribution of GMP-140 over broad expanses of the plasma membrane using surface replication techniques. Fixed platelets were allowed to settle onto poly-L-lysine-coated coverslips and immunolabeled with polyclonal anti-GMP-140, followed by protein A gold. After critical-point drying, rotary-shadowed surface replicas were made. GMP-140 was not present on the surfaces of unstimulated platelets, but thrombin stimulation resulted in the massive expression of GMP-140 on the cell surface, with the immunogold label monodispersed. In contrast, we recently found that GPIIb-IIIa, the fibrinogen receptor, is monodispersed on unstimulated platelets and clustered on activated platelets. Although GMP-140's hemostatic function is unknown, its monodispersed surface pattern implies significant differences form GPIIb-IIIa with respect to ligand binding and/or cytoskeletal interaction.     

Evaluation of platelet function by flow cytometry

Platelet function in whole blood can be comprehensively evaluated by flow cytometry. Flow cytometry can be used to measure platelet reactivity, circulating activated platelets, platelet-platelet aggregates, leukocyte-platelet aggregates, procoagulant platelet-derived microparticles, and calcium flux. Clinical applications of whole blood flow cytometric assays of platelet function in disease states (e.g., acute coronary syndromes, angioplasty, and stroke) may include identification of patients who would benefit from additional antiplatelet therapy and prediction of ischemic events. Circulating monocyte-platelet aggregates appear to be a more sensitive marker of in vivo platelet activation than circulating P-selectin-positive platelets. Flow cytometry can also be used in the following clinical settings: monitoring of GPIIb-IIIa antagonist therapy, diagnosis of inherited deficiencies of platelet surface glycoproteins, diagnosis of storage pool disease, diagnosis of heparin-induced thrombocytopenia, and measurement of the rate of thrombopoiesis.     

细胞骨架对凝血酶诱导人血小板分泌反应的调节(英文)

本文利用血小板表面外露的GMP-140为血小板分泌反应的特异性标志,通过放射免疫分析法定量测定血小板表面GMP-140分子数,研究了细胞骨架抑制剂对凝血酶诱导血小板分泌反应的影响。结果表明,凝血酶激活使血小板表面GMP-140的外露明显增加,反应迅速,并在一定范围内呈剂量和时间依赖性;而ADP刺激则几乎不引起GMP-140外露的增加。凝血酶激活前加入不同的细胞骨架抑制剂处理可产生不同的效应:细胞松驰素B(肌动蛋白微丝抑制剂)可明显上调凝血酶诱导的GMP-140外露;而秋水仙素(微管抑制剂)则下调GMP-140的外露;两者同时处理仍呈现明显的上调作用。提示凝血酶作为一种强激活剂,不仅可通过受体-G蛋白-第二信使的途径启动血小板分泌反应,而且可能经诱导肌动蛋白微丝的形成对分泌反应起反馈性负调节作用。微管的存在则可能对凝血酶诱导的分泌反应起促进作用。虽然两种细胞骨架的作用相反,但以微丝的作用为主,两者间无相互拮抗现象。     

Properties of GMP-140, an inducible granule membrane protein of platelets and endothelium.

GMP-140 is an integral membrane glycoprotein with an apparent Mr of 140,000 located in secretory granules of human platelets and endothelial cells. When these cells are stimulated, the protein is rapidly redistributed to the plasma membrane; therefore, monoclonal antibodies to GMP-140 are useful markers of activated platelets and endothelium. GMP-140 is cysteine-rich and heavily glycosylated. The cDNA-derived amino acid sequence indicates that it contains a number of modular domains that are likely to fold independently. Beginning at the N-terminus, these comprise a "lectin" domain, an "EGF" domain, nine tandem consensus repeats similar to those in complement-binding proteins, a transmembrane domain, and a cytoplasmic tail. Some cDNAs also predict variant forms of GMP-140, including a putative soluble form lacking the transmembrane domain that appears to arise from alternative splicing of mRNA. The domain organization of GMP-140 is strikingly similar to two other vascular cell surface structures: ELAM-1, a cytokine-inducible endothelial cell receptor that binds neutrophils, and a lymphocyte-homing receptor that mediates the adherence of lymphocytes to high endothelial venules of peripheral lymph nodes. These "selectins" constitute a new gene family of receptors with related structure and potentially related function.     

Enhancement by IL-1 beta and IFN-gamma of platelet activation: adhesion to leukocytes via GMP-140/PADGEM protein (CD62).

We have examined the effect of inflammatory cytokines on the platelet activation. IL-1 beta and IFN-gamma were found to enhance the adhesion of thrombin-treated platelets to monocytic leukemia cells (U937), when the adhesion was assayed by platelet-mediated cell agglutination. The agglutination was inhibited by a monoclonal anti-GMP140 antibody or EDTA, suggesting that the enhanced platelet adhesion to the leukemic cells was mediated by GMP140. In addition, these cytokines also increased the release of 5-HT from platelets in the presence of a low concentration of thrombin. These data suggest that platelet functions are regulated by the cytokines and that activated platelets participate in inflammatory process.     

The selectin GMP-140 binds to sialylated, fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells

Granule membrane protein-140 (GMP-140) is an inducible receptor for myeloid leukocytes on activated platelets and endothelium. Like other selectins, GMP-140 recognizes specific oligosaccharide ligands. However, prior data on the nature of these ligands are contradictory. We investigated the structural features required for ligand interaction with GMP-140 using purified GMP-140, cells naturally expressing specific oligosaccharides, and cells expressing cloned glycosyltransferases. Like the related selectin endothelial leukocyte adhesion molecule-1 (ELAM-1), GMP-140 recognizes alpha(2-3)sialylated, alpha(1-3)fucosylated lactosaminoglycans on both myeloid and nonmyeloid cells, including the sequence Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3)GlcNac beta-R (sialyl Lewis x). Recognition requires sialic acid, because cells expressing large amounts of Lewis x, but not sialyl Lewis x, do not interact with GMP-140. Although sialyl Lewis x is expressed by both myeloid HL-60 cells and CHO cells transfected with an alpha 1-3/4 fucosyltransferase, GMP-140 binds with significantly higher affinity to HL-60 cells. Thus, the sialyl Lewis x tetrasaccharide may require additional structural modifications or specific presentations in order for leukocytes in flowing blood to interact rapidly and with high affinity to GMP-140 on activated platelets or endothelium.     

Detection and characterization of monoclonal antibodies to platelet membrane proteins

We have devised a solid-phase radioimmunoassay for the detection and characterization of monoclonal antibodies directed against platelet surface antigens. Platelet membrane proteins, solubilized with 0.1% Triton X-100, were covalently coupled to cyanogen bromide (CNBr)-activated filter paper disks that were than used as the support in antibody binding assays. SDS PAGE of solubilized membrane proteins taken immediately before and after incubation with activated disks indicated that representative amounts of each membrane protein were bound to the disks. Either monoclonal or heterologous anti-platelet antibody could be detected on disks that had been prepared using as little as 50 micrograms of membrane protein per 100 disks. For the detection of antibody, disks were incubated with test sera for 2 h, washed, and incubated with 125I-labeled anti-immunoglobulin G, and the amount of bound radioactivity was determined. The sensitivity of the disk assay in detecting monoclonal antibodies was far greater than that of a corresponding radioimmunoassay that used whole platelets as the solid phase. By linking other proteins such as fibrinogen or anti-mouse subclass specific antisera to CNBr-activated disks, the method was adapted for antibody characterization. The sensitivity and ease with which the assay can be performed make this technique most suitable for screening and characterizing monoclonal antibodies.     

Platelet activation may explain the storage lesion in platelet concentrates

While the exact nature of the dysfunction of stored platelets is not known, it is generally agreed that the platelet's metabolic activity with lactate accumulation presents a significant impediment to prolonged storage. There is an increasing body of evidence that stored platelets have become activated in the preparation and handling of platelet concentrates. Changes in platelet function and structure in concentrates can be explained in terms of sequelae of activation, especially heightened metabolic activity and activation-specific changes in surface glycoproteins on stored platelets. With the use of inhibitors of platelet activation in the preparation of platelet concentrates, the loss of platelet function and integrity is less rapid and platelet metabolic rate is decreased during an extended storage period. Surface levels of glycoprotein Ib, normally decreased during prolonged storage of platelets, are well-preserved in the presence of activation inhibitors. When the use of inhibitors is combined with replacement of plasma with an artificial medium, platelets stored for up to 20 days appear to be metabolically and structurally intact and responsive to stimuli. In summary, platelet activation appears to play a major role in the generation of the storage lesion in platelet concentrates.     

双专－性抗体介导的纤溶作用

用双功能团试剂将抗尿激酶单克隆抗体Ｎ３４的ＩｇＧ和抗人活化血小板α－颗粒膜蛋白ＧＭＰ－１４０单克隆抗体ＳＺ－５１的Ｆａｂ片段通过二硫键共价偶联,偶联的抗体保留了对各自抗原的亲和性。这种对尿激酶和血栓同时具有亲和活性的双专一性抗体（Ｎ３４－ＳＺ－５１）提高低分子量尿激酶的溶栓效率３８倍,且对血浆中纤维蛋白原的含量基本上不影响。