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The thrombospondin (TSP) family of extracellular glycoproteins consists of five members in vertebrates, TSP1 to -4 and TSP5/cartilage oligomeric matrix protein, and a single member in Drosophila. TSPs are modular multimeric proteins. The C-terminal end of a monomer consists of 3-6 EGF-like modules; seven tandem 23-, 36-, or 38-residue aspartate-rich, Ca(2+)-binding repeats; and an approximately 230-residue C-terminal sequence. The Ca(2+)-binding repeats and C-terminal sequence are spaced almost exactly the same in different TSPs and share many blocks of identical residues. We studied the C-terminal portion of human TSP2 from the third EGF-like module through the end of the protein (E3CaG2). E3CaG2, CaG2 lacking the EGF module, and Ca2 composed of only the Ca(2+)-binding repeats were expressed using recombinant baculoviruses and purified from conditioned media of insect cells. As previously described for intact TSP1, E3CaG2 bound Ca(2+) in a cooperative manner as assessed by equilibrium dialysis, and its circular dichroism spectrum was sensitive to the presence of Ca(2+). Mass spectrometry of the recombinant proteins digested with endoproteinase Asp-N revealed that disulfide pairing of the 18 cysteines in the Ca(2+)-binding repeats and C-terminal sequence is sequential, i.e. a 1-2, 3-4, 5-6, etc., pattern.

Thrombospondin 1 (TSP1) is a homotrimeric glycoprotein composed of 150-kDa subunits connected by disulfide bridges. The procollagen module of thrombospondin 1 has been implicated in antiangiogenic activity. Procollagen modules are found in a number of extracellular proteins and are identifiable by 10 cysteines with characteristic spacing. We expressed and studied the procollagen module (C) of human TSP1, both by itself and in the context of the adjoining oligomerization sequence (o) and N-terminal module (N). The coding sequences were introduced into baculoviruses along with an N-terminal signal sequence and C-terminal polyhistidine tag. Proteins were purified from conditioned medium of infected insect cells by nickel-chelate chromatography. NoC is a disulfide bonded trimer and cleaves readily at a site of preferential proteolysis to yield monomeric N and trimeric oC. These are known properties of full-length TSP1. Mass spectroscopy indicated that C is N-glycosylated, and all 10 cysteine residues of C are in disulfides. By equilibrium ultracentrifugation, C is a monomer in physiological salt solution. Circular dichroism, intrinsic fluorescence, and differential scanning calorimetry experiments suggest that the stability of C is determined by the disulfides. The two tryptophans of C are in a polar, exposed environment as assessed by iodide fluorescence quenching and solvent perturbation. The oC far UV circular dichroism spectrum could be modeled as the sum of C and a coiled-coil oligomerization domain. The results indicate that the recombinant C folds autonomously into its native structure, and trimerization of the modules in TSP1 does not perturb their structures.

The integrin alpha 2 beta 1 is a receptor for collagen that plays a fundamental role in the adhesion of blood platelets to the extracellular matrix. We previously reported that platelet alpha 2 beta 1 levels among randomly selected individuals can vary up to 10-fold and that this correlates with differences in adhesiveness to type-I or type-III collagens. We have now found two linked, allelic polymorphisms within the coding sequence of the alpha 2 gene that correlate with receptor density, TTT/TTC at codon Phe224 and ACA/ACG at codon Thr246. By Southern blot hybridization of specific antisense DNA probes to segments of genomic DNA that encompass each coding region, we have determined the gene frequencies of each allele in a random donor population (n = 65) to be 0.585 (TTC...ACG) and 0.415 (TTT...ACA). There is a statistically significant correlation between the alleles TTT...ACA (codons 224...246) and high receptor density (n = 30; P < .002), whereas the complimentary alleles TTC...ACG are associated with low receptor density. Heterozygous individuals express intermediate levels of this receptor, and familial studies confirm that these allelic polymorphisms are inherited characteristics. These findings prove that the level of platelet alpha 2 beta 1 is an inherited trait. The molecular basis for receptor density remains to be determined, but our findings establish that these silent alleles within the coding sequence of the alpha 2 gene are linked to the genetic basis for variation in receptor density.

The Arg-Gly-Asp (RGD) sequence within the third complementarity-determining region (CDR3) of the heavy chain (H3) is responsible for the binding of the recombinant murine Fab molecules, AP7 and PAC1.1, to the platelet integrin alphaIIbbeta3. AP7 binding is minimally influenced by the conformational state of this receptor, whereas PAC1.1 binds preferentially to the activated state of the receptor induced by platelet agonists. To study the molecular basis for this functional difference, we replaced the AP7 H3 loop (HPFYRGDGGN) with all or segments of the analogous sequence from PAC1.1 (RSPSYYRGDGAGP). AP7 Fd (VH domain + Cgamma1 domain) segments containing these H3 loop sequences were expressed as active Fab molecules by coinfection of Spodoptera frugiperda cell lines with recombinant baculoviruses containing Fd and AP7 kappa chain cDNA. Replacement of the entire AP7 H3 loop with that from PAC1.1 generated the mutant AP7.3 Fab molecule, which bound selectively to either activated, gel-filtered platelets or to purified alphaIIbbeta3 in a manner identical to that of PAC1.1. Identical results were obtained when solely the sequences flanking the amino side of RGD within the respective H3 loops were exchanged. AP7.3 and PAC1.1 exhibited saturable but submaximal binding to activated gel-filtered platelets. Relative to AP7, the number of AP7.3 or PAC1. 1 Fab molecules bound per platelet was 17% in the presence of 1 m Ca2+ + 1 mM Mg2+ or 40% in the presence of 10 microM Mn2+. The ratio of Fab molecules bound after versus before activation (mean =/- S.D.; n = 3) was: for AP7.3, 9.8 =/- 0.6; for PAC1.1, 8.8 +/- 0.3; and for AP7, 1.4 =/- 0.2. In addition, AP7 bound to the stably expressed integrin mutant alphaIIbbeta3(S123A), whereas AP7.3 and PAC1 did not. Because AP7.3 behaves in every respect like PAC1.1, we conclude that the ability of RGD-based ligands to distinguish activated from resting conformations of the integrin alphaIIbbeta3 can be regulated by limited amino acid sequences immediately adjacent to the RGD tripeptide. Furthermore, those Fab molecules that exhibit increased selectivity for the activated conformation of alphaIIbbeta3 bind to a subpopulation of this integrin on platelets that is modulated by divalent cations.

Calcium-activated neutral proteinase (calpain) has been shown to cleave proteins involved in the maintenance of cell structure. In human platelets, substrates of calpain include glycoprotein Ib (GPIb), actin-binding protein (ABP), and talin. GPIb-ABP complexes can be isolated in detergent extracts and are thought to represent membrane-cytoskeleton attachment sites. It has been hypothesized that the hydrolysis of GPIb-ABP by calpain is regulated by the extent of binding of this proteinase to the plasma membrane-cytoskeleton interface with platelet activation. Recently, another calpain substrate (talin) has been shown to redistribute from the cytoplasm to the plasma membrane-cytoskeleton interface as the result of thrombin stimulation. To investigate the intracellular distribution of calpain I, we employed the monoclonal antibody B27D8, specific for the heavy chain (catalytic subunit) of calpain I. Indirect immunofluorescent staining of resting human platelets revealed undetectable surface antigen. Permeabilization with Triton X-100, however, revealed a diffuse intracellular antigen consistent with a cytosolic distribution. To determine whether this antigen distribution reflected the proenzyme or the activated form of calpain I and to assess the degree of hydrolysis of ABP, GPIb, and talin, we employed B27D8 and murine monoclonal antibodies against ABP (1B3 and 3D1), GPIb (LJIb10), and rabbit polyclonal antibodies against talin (A2 and B11) in a quantitative immunotransblot assay. Examination of resting platelets revealed that calpain I existed as the 85-kd proenzyme form and that ABP, GPIb, and talin existed in their native intact forms. When platelets were aggregated with thrombin, autoproteolysis of calpain I occurred within the 30 seconds required to completely solubilize platelet aggregates in sodium dodecyl sulfate-containing buffer and not as a direct result of thrombin-induced activation.(ABSTRACT TRUNCATED AT 250 WORDS)

The platelet glycoprotein (GP) IIb-IIIa complex figures prominently as an immunogen in autoimmune (idiopathic) thrombocytopenic purpura (ITP). 2E7 is a human monoclonal IgM autoantibody, derived from splenocytes of a patient with ITP, that recognizes a specific octapeptide amino acid sequence, Phe-Asp-Gly-Tyr-Trp-Gly-Tyr-Ser, on the heavy chain of GPIIb. This represents the first precise identification of an epitope on GPIIb-IIIa recognized by a human antibody. In this study, we have isolated total mRNA from 2E7, synthesized the corresponding cDNA using reverse transcriptase, and amplified the immunoglobulin mu and kappa chain cDNA by the Taq 1 polymerase chain reaction (PCR) using specific primers. The 2E7 mu chain variable region is encoded by a VH3 gene segment that is 98% homologous to the germline gene VH1.9III, a D-gene that is not homologous to any of the germline D-genes reported to date, and a JH6 gene segment that is essentially germline. The heavy-chain sequence, save for the unique D-gene, is similar to that of a number of human autoantibodies. The 2E7 kappa variable region is encoded by a Vk1 gene segment linked to a Jk1 gene segment. The Vk1 sequence of 2E7, with the exception of one nucleotide, is identical to that of autoantibody HF2-1/17, a prototype of SLE-associated anti-DNA autoantibodies bearing the 16/6 idiotype. The single base substitution results in a relatively conservative exchange of Asp for Glu at position 70 of the protein sequence. Despite this near identity in sequence, 2E7 does not bind to either single-stranded or double-stranded DNA. From these results, we conclude that specificity of 2E7 is likely to reside in either or both the D-JH region (CDR3) of the mu chain and the Jk region (CDR3) of the kappa chain. In addition to the identification of a novel D-gene, we also provide evidence that the 2E7 VHIII gene is probably a prototype of a VHIII subfamily, that the germline Vk1 gene shared by 2E7 and autoantibodies of the 16/6 idiotype probably represents a separate Vk family, and that this Vk gene cannot itself attribute specificity for DNA.