Samuel A. Santoro

Antiphospholipid Antibodies: Anticardiolipin and the Lupus Anticoagulant in Systemic Lupus Erythematosus (SLE) and in Non-SLE Disorders: Prevalence and Clinical Significance

PURPOSE To determine the prevalence of lupus anticoagulant and anticardiolipin in systemic lupus erythematosus (SLE) and in non-SLE disorders, and to evaluate the clinical significance of these autoantibodies as they relate to thromboembolic events, neuropsychiatric disorders, thrombocytopenia, and fetal loss. DATA IDENTIFICATION A computer-assisted search of the literature (MEDLINE, 1966 to 1989) and review of the bibliographies of all identified articles. STUDY SELECTION Series of ten or more subjects were included if the assays used for detecting lupus anticoagulant or anticardiolipin met the specified minimal criteria for validity. DATA EXTRACTION Series were categorized according to antibody type and underlying disease. A systematic appraisal of patient selection methods, study design, and assay methods was done. RESULTS OF DATA ANALYSIS An analysis of 29 published series (comprising over 1000 patients with SLE) yielded an average frequency of 34% for the lupus anticoagulant and 44% for anticardiolipin. Antiphospholipid antibodies are also prevalent in patients with various non-SLE disorders. In patients with SLE, a statistically significant association exists between the presence of either antibody and a history of thrombosis, neurologic disorders, or thrombocytopenia. The available data suggest, but do not firmly support, an association between antiphospholipid antibodies and history of fetal loss in women with SLE. Contrary to prevailing opinion, none of these associations have been shown conclusively in patients with non-SLE disorders. CONCLUSIONS The results of predominantly retrospective series suggest that for certain persons (patients with SLE or closely related disorders) antiphospholipid antibodies may be important risk factors for thrombosis, neurologic disease, thrombocytopenia, and fetal loss. Standardized tests for lupus anticoagulant and anticardiolipin, as well as long-term, prospective clinical studies, are needed to determine the prognostic value of antiphospholipid antibodies.

Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen

Platelets initially adhere to collagen via a divalent cation-dependent process supported by Mg2+, Mn2+, Fe2+, Cu2+, Zn2+, or Co2+ more rapidly and to a greater extent than by previously studied divalent cation-independent mechanisms. Ca2+ not only fails to support adhesion, it is inhibitory. Platelet activation and secretion are not required for adhesion by this mechanism. Monomeric and fibrillar collagens, but not denatured collagen, effectively support divalent cation-dependent adhesion. Types I, III, and IV collagen, but not type V collagen, support adhesion. A platelet surface protein of Mr 160,000, possibly identical with platelet membrane glycoprotein Ia, that binds to collagen with the appropriate divalent cation specificity has been identified and is the likely mediator of the initial divalent cation-dependent adhesion of platelets to collagen.

The α2 Integrin Subunit-Deficient Mouse: A Multifaceted Phenotype Including Defects of Branching Morphogenesis and Hemostasis

The alpha(2)beta(1) integrin is a collagen/laminin receptor expressed on platelets, endothelial cells, fibroblasts, and epithelial cells. To define the role of the alpha(2)beta(1) integrin in vivo, we created a genetically engineered mouse in which expression of the alpha(2)beta(1) integrin was completely eliminated. Mice deficient in the alpha(2)beta(1) integrin are viable, fertile, and develop normally with no excess lethality of homozygotes. Both alpha(2)beta(1)-integrin protein and alpha(2) mRNA were undetectable in the alpha(2)-null mice. Gross and histological evaluation of the heart, lungs, kidneys, gastrointestinal tract, pancreas, skin, and reproductive tracts revealed no abnormalities. However, quantitative analysis of mammary gland branching morphogenesis demonstrated that branching complexity is markedly diminished in the alpha(2)-deficient animals. Studies in the alpha(2)-deficient animals do not support the proposed roles for the alpha(2)beta(1) integrin on fibroblasts and keratinocytes in wound healing. When compared to platelets from wild-type littermates, platelets from alpha(2)-null mice failed to adhere to type I collagen under either static or shear-stress conditions. Although platelets from alpha(2)-deficient animals aggregated in response to collagen, they did so with prolonged lag time and lessened intensity. The alpha(2)beta(1) integrin-null mouse thus exhibits diverse, sometimes subtle, phenotypes consistent with the widespread pattern of alpha(2)beta(1) integrin expression.

The impact of heparin concentration and activated clotting time monitoring on blood conservation: A prospective, randomized evaluation in patients undergoing cardiac operation*

A whole blood hemostasis system (Hepcon) provides both activated clotting time and accurate whole blood heparin concentration measurements via an automated protamine titration method. This study was designed to prospectively evaluate the impact of heparin and protamine administration using this system on the incidence and treatment of bleeding after cardiopulmonary bypass. Two hundred fifty-four patients requiring cardiopulmonary bypass were enrolled in this prospective study over a 7-month period. Patients treated with antifibrinolytic agents (aprotinin, epsilon-aminocaproic or tranexamic acid) were excluded. Patients were randomly assigned to either a control (n = 127) or intervention (n = 127) group. For control patients, the anticoagulation protocol consisted of an initial fixed dose of 250 U/kg of heparin, and additional 5000 U heparin doses were administered if the activated clotting time was less than 480 seconds. Heparin was neutralized with an initial fixed dose of protamine (0.8 mg protamine per milligram total heparin). For the intervention group, an initial dose of heparin was based on an automated heparin dose-response assay. Additional heparin doses were administered if the heparin concentration was less than the reference concentration or for an activated clotting time less than 480 seconds. The protamine dose was based on the residual heparin concentration. Treatment of excessive bleeding after cardiopulmonary bypass was based on an algorithm using point-of-care testing with whole blood prothrombin time, activated partial thromboplastin time, heparinase activated clotting time, and platelet count. No differences between the two treatment groups were identified in reference to demographic factors, preoperative anticoagulant medications, preoperative coagulation data, number of reoperations, or combined procedures and duration of cardiopulmonary bypass. Indirect evidence for coagulation factor consumption was demonstrated in control patients by more prolonged whole blood prothrombin time and activated partial thromboplastin time values after cardiopulmonary bypass when compared with values obtained in the intervention group. Patients in the intervention cohort received greater doses of heparin (intervention: 612 +/- 147, control: 462 +/- 114 U/kg, p < 0.0001) and had lower protamine to heparin ratios (intervention: 0.70 +/- 0.64, control: 0.94 +/- 0.21, p = 0.0001) compared with control patients. Patients in the intervention cohort received significantly fewer platelet (intervention: 1.7 +/- 3.6 U, control: 3.7 +/- 6.7 U, p = 0.003), plasma (intervention: 0.4 +/- 1.3 U, control: 1.4 +/- 2.5 U, p = 0.0001), and cryoprecipitate units (intervention: 0.0 +/- 0.0 U, control: 0.2 +/- 1.2 U, p = 0.04) during the perioperative interval than control patients.(ABSTRACT TRUNCATED AT 400 WORDS)

Decorin Binds Near the C Terminus of Type I Collagen

Decorin belongs to a family of small leucine-rich proteoglycans that are directly involved in the control of matrix organization and cell growth. Genetic evidence indicates that decorin is required for the proper assembly of collagenous matrices. Here, we sought to establish the precise binding site of decorin on type I collagen. Using rotary shadowing electron microscopy and photoaffinity labeling, we mapped the binding site of decorin protein core to a narrow region near the C terminus of type I collagen. This region is located within the cyanogen bromide peptide fragment α1(I) CB6 and is ∼25 nm from the C terminus, in a zone that coincides with the c1 band of the collagen fibrild-period. This location is very close to one of the major intermolecular cross-linking sites of collagen heterotrimers. Thus, decorin protein core possesses a unique binding specificity that could potentially regulate collagen fibril stability.

Tumor cell α3β1 integrin and vascular laminin-5 mediate pulmonary arrest and metastasis

Arrest of circulating tumor cells in distant organs is required for hematogenous metastasis, but the tumor cell surface molecules responsible have not been identified. Here, we show that the tumor cell α3β1 integrin makes an important contribution to arrest in the lung and to early colony formation. These analyses indicated that pulmonary arrest does not occur merely due to size restriction, and raised the question of how the tumor cell α3β1 integrin contacts its best-defined ligand, laminin (LN)-5, a basement membrane (BM) component. Further analyses revealed that LN-5 is available to the tumor cell in preexisting patches of exposed BM in the pulmonary vasculature. The early arrest of tumor cells in the pulmonary vasculature through interaction of α3β1 integrin with LN-5 in exposed BM provides both a molecular and a structural basis for cell arrest during pulmonary metastasis.

COMPARISON OF ACTIVATED COAGULATION TIME AND WHOLE BLOOD HEPARIN MEASUREMENTS WITH LABORATORY PLASMA ANTI-XA HEPARIN CONCENTRATION IN PATIENTS HAVING CARDIAC OPERATIONS

Previous reports suggest that activated clotting times do not correlate with heparin concentration during cardiopulmonary bypass. This study was designed to compare whole blood heparin concentration and activated clotting time measurements with laboratory-based plasma heparin concentration. Sixty-two patients having cardiac operations requiring cardiopulmonary bypass were enrolled in this study. The study was conducted in two phases. In phase I of this trial, blood specimens were obtained from 30 patients before heparin administration and after each of three heparin doses (20, 80, and 150 U/kg). In phase II, blood specimens were obtained from 32 patients before heparin administration and 10 minutes after each of the following: heparin administration (250 or 300 U/kg), initiation of cardiopulmonary bypass, achievement of hypothermia, initiation of rewarming, and immediately before discontinuation of bypass. Blood specimens were used to measure activated clotting time (kaolin and celite), whole blood heparin concentration, and anti-factor Xa plasma heparin concentration. In phase I, activated clotting time (celite: r = 0.91; kaolin: r = 0.93) and whole blood heparin concentration (r = 0.98) measurements correlated well with plasma heparin concentration. After initiation of cardiopulmonary bypass (phase II), weak correlations for activated clotting time measurements (celite: r = 0.34; kaolin: r = 0.59) and a strong correlation for whole blood heparin concentration (r = 0.95) were evident when compared with plasma heparin concentration. During bypass, activated clotting time measurements also inversely correlated with temperature (celite: r = -0.21; kaolin: r = -0.19) and hematocrit (celite: r = -0.26; kaolin: r = -0.21). A weak correlation between activated clotting time measurements and plasma heparin concentration is evident during the cardiopulmonary bypass period, probably because of the influence of both reduced hematocrit and temperature on the activated clotting time assay. In contrast, whole blood heparin measurements correlate well with plasma heparin concentration before and during bypass. Further studies are needed to determine whether maintaining heparin levels during cardiopulmonary bypass by monitoring heparin concentration is more effective in preventing consumptive activation of the hemostatic system, reducing bleeding, and minimizing the use of blood products after cardiopulmonary bypass when compared with a protocol based on activated clotting time.

Competition for related but nonidentical binding sites on the glycoprotein IIb-IIIa complex by peptides derived from platelet adhesive proteins

Two distinct sequences of amino acids, RGDS and HHLGGAKQAGDV, each inhibit the binding of fibrinogen, fibronectin, and von Willebrand factor to the platelet membrane glycoprotein IIb-IIIa complex. We have employed radiolabeled, photoactivatable aryl azide derivatives of the two sequences to explore the relationship between the binding sites for these peptides on the glycoprotein IIb-IIIa complex. Each probe specifically labeled only the glycoprotein IIb-IIIa complex of intact platelets. Since each peptide inhibited labeling of the receptor complex by the other, the peptides compete for binding sites on the receptor complex. However, the binding sites do not appear to be identical. Whereas the RGDS probe specifically labeled both glycoproteins IIb and IIIa, the HHLGGAKQA-GDV probe specifically labeled only glycoprotein IIb.

Isolation and characterization of a platelet surface collagen binding complex related to VLA-2.

A heterodimeric, Mg++-dependent, collagen binding protein has been isolated from platelet membranes. Electrophoretic properties and monoclonal antibody reactivity indicate that the heavy chain of the complex is platelet membrane glycoprotein Ia and that the light chain is glycoprotein IIa. Furthermore, the receptor appears to be identical with the recently defined VLA-2 complex found on activated T-lymphocytes, platelets and other cells. When incorporated into liposomes, the purified complex mediates the Mg++-dependent adhesion of the liposomes to collagen substrates. These observations suggest that the VLA-2 complex mediates cellular adhesion to collagen in platelets and possibly in other cells.

β1 Integrin Mediates Internalization of Mammalian Reovirus

ABSTRACT Reovirus infection is initiated by interactions between the attachment protein σ1 and cell surface carbohydrate and junctional adhesion molecule A (JAM-A). Expression of a JAM-A mutant lacking a cytoplasmic tail in nonpermissive cells conferred full susceptibility to reovirus infection, suggesting that cell surface molecules other than JAM-A mediate viral internalization following attachment. The presence of integrin-binding sequences in reovirus outer capsid protein λ2, which serves as the structural base for σ1, suggests that integrins mediate reovirus endocytosis. A β1 integrin-specific antibody, but not antibodies specific for other integrin subunits, inhibited reovirus infection of HeLa cells. Expression of a β1 integrin cDNA, along with a cDNA encoding JAM-A, in nonpermissive chicken embryo fibroblasts conferred susceptibility to reovirus infection. Infectivity of reovirus was significantly reduced in β1-deficient mouse embryonic stem cells in comparison to isogenic cells expressing β1. However, reovirus bound equivalently to cells that differed in levels of β1 expression, suggesting that β1 integrins are involved in a postattachment entry step. Concordantly, uptake of reovirus virions into β1-deficient cells was substantially diminished in comparison to viral uptake into β1-expressing cells. These data provide evidence that β1 integrin facilitates reovirus internalization and suggest that viral entry occurs by interactions of reovirus virions with independent attachment and entry receptors on the cell surface.