Physiologic variations in blood plasminogen levels affect outcomes after acute cerebral thromboembolism in mice: a pathophysiologic role for microvascular thrombosis.

UNLABELLED: Essentials Physiologic variations in blood plasminogen (Pg) levels may affect ischemic stroke outcomes. We tested Pg effects in a model with translational relevance to human thromboembolic stroke. A dose-response exists between Pg levels and brain injury, fibrinolysis, barrier breakdown. Higher Pg levels reduce microvascular thrombosis and improve outcomes in ischemic stroke. SUMMARY: Background and Objectives Plasminogen appears to affect brain inflammation, cell movement, fibrinolysis, neuronal excitotoxicity, and cell death. However, brain tissue and circulating blood plasminogen may have different roles, and there is wide individual variation in blood plasminogen levels. The aim of this study was to determine the integrated effect of blood plasminogen levels on ischemic brain injury. Methods We examined thromboembolic stroke in mice with varying, experimentally determined, blood plasminogen levels. Ischemic brain injury, blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis were determined. Results Within the range of normal variation, plasminogen levels were strongly associated with ischemic brain injury; higher blood plasminogen levels had dose-related, protective effects. Higher plasminogen levels were associated with increased dissolution of the middle cerebral artery thrombus. Higher plasminogen levels decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression and microvascular thrombosis in the ischemic brain. In plasminogen-deficient mice, selective restoration of blood plasminogen levels reversed the harmful effects of plasminogen deficiency on ischemic brain injury. Specific inhibition of thrombin also reversed the effect of plasminogen deficiency on ischemic injury by decreasing microvascular thrombosis, blood-brain barrier breakdown, and matrix metalloproteinase-9 expression. Conclusions Variation in blood plasminogen levels, within the range seen in normal individuals, had marked effects on experimental ischemic brain injury. Higher plasminogen levels protected against ischemic brain injury, and decreased blood-brain barrier breakdown, matrix metalloproteinase-9 expression, and microvascular thrombosis. The protective effects of blood plasminogen appear to be mediated largely through a decrease in microvascular thrombosis in the ischemic territory.

Reprogrammed streptokinases develop fibrin-targeting and dissolve blood clots with more potency than tissue plasminogen activator.

BACKGROUND: Given the worldwide epidemic of cardiovascular diseases, a more effective means of dissolving thrombi that cause heart attacks, could markedly reduce death, disability and healthcare costs. Plasminogen activators (PAs) such as streptokinase (SK) and tissue plasminogen activator (TPA) are currently used to dissolve fibrin thrombi. SK is cheaper and more widely available, but it appears less effective because it lacks TPA's fibrin-targeted properties that focus plasminogen activation on the fibrin surface. OBJECTIVE: We examined whether re-programming SK's mechanism of action would create PAs with greater fibrin-targeting and potency than TPA. METHODS AND RESULTS: When fibrinogen consumption was measured in human plasma, reprogrammed molecules SKDelta1 and SKDelta59 were 5-fold and > 119-fold more fibrin-dependent than SK (P < 0.0001), and 2-fold and > 50-fold more fibrin-dependent than TPA (P < 0.001). The marked fibrin-targeting of SKDelta59 was due to the fact that: (i) it did not generate plasmin in plasma, (ii) it was rapidly inhibited by alpha2-antiplasmin, and (iii) it only processed fibrin-bound plasminogen. To assess the fibrin-targeting and therapeutic potential of these PAs in vivo, a novel 'humanized' fibrinolysis model was created by reconstituting plasminogen-deficient mice with human plasminogen. When compared with TPA, SKDelta1 and SKDelta59 were 4-fold (P < 0.0001) and 2-fold (P < 0.003) more potent at dissolving blood clots in vivo, respectively, on a mass-dose basis and 2-3 logs more potent than TPA (P < 0.0001) when doses were calibrated by standard activity assays. CONCLUSION: These experiments suggest that reprogramming SK's mechanism of action markedly enhances fibrin-targeting and creates, in comparison with TPA, activators with greater fibrinolytic potency.

Fibrinolysis is amplified by converting alpha-antiplasmin from a plasmin inhibitor to a substrate.

alpha(2)-Antiplasmin (alpha(2)-AP) is the fast serpin inhibitor of plasmin and appears to limit the success of treatment for thrombosis. We examined the mechanisms through which monoclonal antibodies (mAbs) against alpha(2)-AP amplify fibrinolysis. The mAbs RWR, 49 and 77 interfered with the ability of alpha(2)-AP to inhibit plasmin, microplasmin and trypsin. In solution, mAbs 49 and 77 bound to alpha(2)-AP with 5-fold to 10-fold higher relative affinity than mAb-RWR, while mAb-RWR bound with greater avidity to immobilized or denatured alpha(2)-AP. Binding studies with chimeric alpha(2)-APs revealed that none of the mAbs bound to sites in alpha(2)-AP that form putative contacts with plasmin, namely the carboxy terminal lysines of alpha(2)-AP, or the reactive center loop in the serpin domain of alpha(2)-AP. Rather, mAb-RWR recognized an epitope in the amino-terminus of alpha(2)-AP (L(13)GNQEPGGQTALKSPPGVCS(32)) near the site at which alpha(2)-AP cross-links to fibrin. mAbs 49 and 77 bound to another conformational epitope in the serpin domain of alpha(2)-AP. mAbs 49 and 77 markedly increased the stoichiometry of plasmin inhibition by alpha(2)-AP (from 1.1 +/- 0.1 to 51 +/- 4 and 67 +/- 7) indicating that they convert alpha(2)-AP from an inhibitor to a substrate of plasmin. This was confirmed by sodium dodecylsulfate polyacrylamide gel electrophoresis analysis showing cleavage of alpha(2)-AP by plasmin in the presence of these mAbs. In summary, these mAbs appear to act at sites distinct from known alpha(2)-AP-plasmin contacts to enhance fibrinolysis by converting alpha(2)-AP from an inhibitor to a plasmin substrate.

The mechanism of a bacterial plasminogen activator intermediate between streptokinase and staphylokinase.

The therapeutic properties of plasminogen activators are dictated by their mechanism of action. Unlike staphylokinase, a single domain protein, streptokinase, a 3-domain (alpha, beta, and gamma) molecule, nonproteolytically activates human (h)-plasminogen and protects plasmin from inactivation by alpha(2)-antiplasmin. Because a streptokinase-like mechanism was hypothesized to require the streptokinase gamma-domain, we examined the mechanism of action of a novel two-domain (alpha,beta) Streptococcus uberis plasminogen activator (SUPA). Under conditions that quench trace plasmin, SUPA nonproteolytically generated an active site in bovine (b)-plasminogen. SUPA also competitively inhibited the inactivation of plasmin by alpha(2)-antiplasmin. Still, the lag phase in active site generation and plasminogen activation by SUPA was at least 5-fold longer than that of streptokinase. Recombinant streptokinase gamma-domain bound to the b-plasminogen.SUPA complex and significantly reduced these lag phases. The SUPA-b.plasmin complex activated b-plasminogen with kinetic parameters comparable to those of streptokinase for h-plasminogen. The SUPA-b.plasmin complex also activated h-plasminogen but with a lower k(cat) (25-fold) and k(cat)/K(m) (7.9-fold) than SK. We conclude that a gamma-domain is not required for a streptokinase-like activation of b-plasminogen. However, the streptokinase gamma-domain enhances the rates of active site formation in b-plasminogen and this enhancing effect may be required for efficient activation of plasminogen from other species.

Catalytic life of activated factor XIII in thrombi. Implications for fibrinolytic resistance and thrombus aging.

BACKGROUND: Because the increased fibrinolytic resistance of older thrombi may be caused by the continuous cross-linking action of fibrin-bound activated factor XIII (FXIIIa), we examined the persistence of FXIIIa catalytic activity in clots of various ages. METHODS AND RESULTS: The time-related changes in FXIIIa activity in clots was measured with (1) alpha(2)-antiplasmin (alpha(2)AP), a physiological glutamine substrate; (2) alpha(2)AP(13-24), a peptide; and (3) pentylamine, a nonspecific lysine substrate. The cross-linking of alpha(2)AP, alpha(2)AP(13-24), and pentylamine into fibrin by clot-bound FXIIIa declined rapidly with half-lives of 19, 21, and 26 minutes, respectively. Mutational studies showed that glutamine 14 (but not glutamine 3 or 16) and valine 17 of alpha(2)AP(13-24) were required for efficient cross-linking to fibrin. The loss of activity was not due primarily to FXIIIa proteolysis and was partially restored by reducing agents, suggesting that oxidation contributes to the loss of the enzyme's activity in clots. In vivo, the ability of thrombus-bound FXIIIa to cross-link an infused alpha(2)AP(13-24) peptide into existing pulmonary emboli also declined significantly over time. CONCLUSIONS: FXIIIa cross-links alpha(2)AP and an alpha(2)AP peptide, in a sequence-specific manner, into formed clots with a catalytic half-life of approximately 20 minutes. This indicates that FXIIIa activity is a hallmark of new thrombi and that the antifibrinolytic cross-linking effects of FXIIIa are achieved more rapidly in thrombi than previously believed.

Leucine 42 in the fibronectin motif of streptokinase plays a critical role in fibrin-independent plasminogen activation.

The NH(2) terminus (residues 1-59) of streptokinase (SK) is a molecular switch that permits fibrin-independent plasminogen activation. Targeted mutations were made in recombinant (r) SK1-59 to identify structural interactions required for this process. Mutagenesis established the functional roles of Phe-37and Glu-39, which were projected to interact with microplasmin in the activator complex. Mutation of Leu-42 (rSK1-59(L42A)), a conserved residue in the SK fibronectin motif that lacks interactions with microplasmin, strongly reduced plasminogen activation (k(cat) decreased 50-fold) but not amidolysis (k(cat) decreased 1.5-fold). Otherwise rSK1-59(L42A) and native rSK1-59 were indistinguishable in several parameters. Both displayed saturable and specific binding to Glu-plasminogen or the remaining SK fragment (rSKDelta59). Similarly rSK1-59 and rSK1-59(L42A) bound simultaneously to two different plasminogen molecules, indicating that both plasminogen binding sites were intact. However, when bound to SKDelta59, rSK1-59(L42A) was less effective than rSK1-59 in restructuring the native conformation of the SK A domain, as detected by conformation-dependent monoclonal antibodies. In the light of previous studies, these data provide evidence that SK1-59 contributes to fibrin-independent plasminogen activation through 1) intermolecular interactions with the plasmin in the activator complex, 2) binding interactions with the plasminogen substrate, and 3) intramolecular interactions that structure the A domain of SK for Pg substrate processing.

Epsilon amino caproic acid inhibits streptokinase-plasminogen activator complex formation and substrate binding through kringle-dependent mechanisms.

Lysine side chains induce conformational changes in plasminogen (Pg) that regulate the process of fibrinolysis or blood clot dissolution. A lysine side-chain mimic, epsilon amino caproic acid (EACA), enhances the activation of Pg by urinary-type and tissue-type Pg activators but inhibits Pg activation induced by streptokinase (SK). Our studies of the mechanism of this inhibition revealed that EACA (IC(50) 10 microM) also potently blocked amidolytic activity by SK and Pg at doses nearly 10000-fold lower than that required to inhibit the amidolytic activity of plasmin. Different Pg fragments were used to assess the role of the kringles in mediating the inhibitory effects of EACA: mini-Pg which lacks kringles 1-4 of Glu-Pg and micro-Pg which lacks all kringles and contains only the catalytic domain. SK bound with similar affinities to Glu-Pg (K(A) = 2.3 x 10(9) M(-1)) and to mini-Pg (K(A) = 3.8 x 10(9) M(-)(1)) but with significantly lower affinity to micro-Pg (K(A) = 6 x 10(7) M(-)(1)). EACA potently inhibited the binding of Glu-Pg to SK (K(i) = 5.7 microM), but was less potent (K(i) = 81.1 microM) for inhibiting the binding of mini-Pg to SK and had no significant inhibitory effects on the binding of micro-Pg and SK. In assays simulating substrate binding, EACA also potently inhibited the binding of Glu-Pg to the SK-Glu-Pg activator complex, but had negligible effects on micro-Pg binding. Taken together, these studies indicate that EACA inhibits Pg activation by blocking activator complex formation and substrate binding, through a kringle-dependent mechanism. Thus, in addition to interactions between SK and the protease domain, interactions between SK and the kringle domain(s) play a key role in Pg activation.

A catalytic switch and the conversion of streptokinase to a fibrin-targeted plasminogen activator.

Plasminogen (Pg) activators such as streptokinase (SK) save lives by generating plasmin to dissolve blood clots. Some believe that the unique ability of SK to activate Pg in the absence of fibrin limits its therapeutic utility. We have found that SK contains an unusual NH(2)-terminal "catalytic switch" that allows Pg activation through both fibrin-independent and fibrin-dependent mechanisms. Unlike SK, a mutant (rSKDelta59) fusion protein lacking the 59 NH(2)-terminal residues was no longer capable of fibrin-independent Pg activation (k(cat)/K(m) decreased by >600-fold). This activity was restored by coincubation with equimolar amounts of the NH(2)-terminal peptide rSK1-59. Deletion of the NH(2) terminus made rSKDelta59 a Pg activator that requires fibrin, but not fibrinogen, for efficient catalytic function. The fibrin-dependence of the rSKDelta59 activator complex apparently resulted from selective catalytic processing of fibrin-bound Pg substrates in preference to other Pg forms. Consistent with these observations, the presence (rSK) or absence (rSKDelta59) of the SK NH(2)-terminal peptide markedly altered fibrinolysis of human clots suspended in plasma. Like native SK, rSK produced incomplete clot lysis and complete destruction of plasma fibrinogen; in contrast, rSKDelta59 produced total clot lysis and minimal fibrinogen degradation. These studies indicate that structural elements in the NH(2) terminus are responsible for SK's unique mechanism of fibrin-independent Pg activation. Because deletion of the NH(2) terminus alters SK's mechanism of action and targets Pg activation to fibrin, there is the potential to improve SK's therapeutic efficacy.

Human platelets contain SNARE proteins and a Sec1p homologue that interacts with syntaxin 4 and is phosphorylated after thrombin activation: implications for platelet secretion.

In response to thrombin and other extracellular activators, platelets secrete molecules from large intracellular vesicles (granules) to initiate thrombosis. Little is known about the molecular machinery responsible for vesicle docking and secretion in platelets and the linkage of that machinery to cell activation. We found that platelet membranes contain a full complement of interacting proteins-VAMP, SNAP-25, and syntaxin 4-that are necessary for vesicle docking and fusion with the plasma membrane. Platelets also contain an uncharacterized homologue of the Sec1p family that appears to regulate vesicle docking through its binding with a cognate syntaxin. This platelet Sec1 protein (PSP) bound to syntaxin 4 and thereby excluded the binding of SNAP-25 with syntaxin 4, an interaction critical to vesicle docking. As predicted by its sequence, PSP was detected predominantly in the platelet cytosol and was phosphorylated in vitro by protein kinase C (PKC), a secretion-linked kinase, incorporating 0.87 +/- 0.11 mol of PO4 per mole of protein. PSP was also specifically phosphorylated in permeabilized platelets after cellular stimulation by phorbol esters or thrombin and this phosphorylation was blocked by the PKC inhibitor Ro-31-8220. Phosphorylation by PKC in vitro inhibited PSP from binding to syntaxin 4. Taken together, these studies indicate that platelets, like neurons and other cells capable of regulated secretion, contain a unique complement of interacting vesicle docking proteins and PSP, a putative regulator of vesicle docking. The PKC-dependent phosphorylation of PSP in activated platelets and its inhibitory effects on syntaxin 4 binding provide a novel functional link that may be important in coupling the processes of cell activation, intracellular signaling, and secretion.

The contribution of activated factor XIII to fibrinolytic resistance in experimental pulmonary embolism.

BACKGROUND: The resistance of thrombi to fibrinolysis induced by plasminogen activators remains a major impediment to the successful treatment of thrombotic diseases. This study examines the contribution of activated factor XIII (factor XIIIa) to fibrinolytic resistance in experimental pulmonary embolism. METHODS AND RESULTS: The fibrinolytic effects of specific inhibitors of factor XIIIa-mediated fibrin-fibrin cross-linking and alpha2-antiplasmin-fibrin cross-linking were measured in anesthetized ferrets with pulmonary emboli. Five experimental groups were treated with heparin (100 U/kg) and/or tissue plasminogen activator (TPA, 1 mg/kg) and the percent (mean+/-SD) lysis of emboli was determined: (1) control, normal factor XIIIa activity (14.1+/-4. 8% lysis); (2) inhibited factor XIIIa activity (42.7+/-7.4%); (3) normal factor XIIIa activity+TPA (32.3+/-7.7%); (4) inhibited factor XIIIa activity+TPA (76.0+/-11.9%); and (5) inhibited alpha2-antiplasmin-fibrin cross-linking+TPA (54.7+/-3.9%). Inhibition of factor XIIIa activity increased endogenous lysis markedly (group 1 versus 2; P<0.0001), to a level comparable to that achieved with TPA (group 2 versus 3; P<0.05). Among groups receiving TPA, selective inhibition of factor XIII-mediated alpha2-antiplasmin-fibrin cross-linking enhanced lysis (group 3 versus 5; P<0.0005). Complete inhibition of factor XIIIa also amplified lysis (group 3 versus 4; P<0.0001) and had greater effects than inhibition of alpha2-antiplasmin cross-linking alone (group 4 versus 5; P<0.0005). No significant fibrinogen degradation occurred in any group. CONCLUSIONS: Factor XIIIa-mediated fibrin-fibrin and alpha2-antiplasmin-fibrin cross-linking both caused experimental pulmonary emboli to resist endogenous and TPA-induced fibrinolysis. This suggests that factor XIIIa may play a critical role in regulating fibrinolysis in human thrombosis.

Comparative biochemical and ultrastructural studies of P-selectin in rabbit platelets.

The role of platelets in thrombotic vascular disease has been widely studied in rabbits. Yet, in rabbit platelets, there is little known about the alpha-granules, which contain many of the key effector molecules for thrombosis. In this comparative study of rabbit platelets, we have characterized the structure and expression of P-selectin, an alpha-granule membrane protein that mediates leukocyte adhesion and thrombus propagation. The sequences of tryptic peptides of rabbit P-selectin show an overall sequence identity of 74% with human P-selectin, and 69-77% identity with cow, dog, mouse, rat and sheep P-selectins. The mean (+/- S.D.) apparent molecular mass of reduced rabbit P-selectin is 117 +/- 7 kDa which is approximately 8 kDa larger than the unreduced protein (109 +/- 5 kDa). Rabbit P-selectin appears smaller than human P-selectin, but is comparable to other species P-selectins, that have fewer 'complement regulatory protein' repeat domains. Cell membrane labeling experiments and antibody binding studies indicate that rabbit P-selectin is nearly absent from the surface of platelets (290 +/- 30 molecules cell-1). However, cellular activation with thrombin causes nearly a 30-fold increase in expression to 14,200 +/- 1100 molecules cell-1. P-selectin is also be expressed on the surface of rabbit platelets activated by other agonists like ADP, A23817 and epinephrine. This selective expression is explained by immunoelectronmicroscopic studies, which show that rabbit P-selectin is sequestered in the intracellular granules of resting platelets. After cell activation by thrombin, P-selectin is found decorating the external membranes of platelet pseudopodia and the surface connected canalicular system. In summary, these studies of P-selectin in rabbit platelets indicate that it is similar in structure, cell localization and expression to human and other species P-selectins. This suggests that studies of P-selectin in thrombosis in rabbits are likely to provide useful insights into the role of this molecule in human thrombotic vascular disease and related conditions.

Alpha 2-antiplasmin causes thrombi to resist fibrinolysis induced by tissue plasminogen activator in experimental pulmonary embolism.

BACKGROUND: In patients with pulmonary embolism, thrombi resist fibrinolysis induced by plasminogen activators. Because the molecular basis of this thrombus resistance is poorly understood, we used a potent inhibitor to examine the potential role of alpha 2-antiplasmin (alpha 2AP) in experimental pulmonary embolism. METHODS AND RESULTS: Lysis of experimental pulmonary emboli was measured 4 hours after embolization in anesthetized ferrets. All animals received heparin (100 U/kg). Five experimental groups were studied: (1) no recombinant tissue plasminogen activator (rTPA); (2) rTPA at 1 mg/kg; (3) rTPA at 2 mg/kg; (4) rTPA at 1 mg/kg plus a control monoclonal antibody (MAb); and (5) rTPA at 1 mg/kg plus an alpha 2AP inhibitor (MAb 77A3). In comparison with ferrets receiving no rTPA (15.6 +/- 10.5% lysis, mean +/- SD), rTPA-treated groups showed significantly greater lysis (P < .01). Animals treated with rTPA and alpha 2AP inhibitor (56.2 +/- 4.7% lysis) showed significantly greater lysis than all other treatment groups, including ferrets treated with the same dose of rTPA alone (38.5 +/- 6.3%, P < .01), with twice the rTPA dose alone (45.0 +/- 6.5%, P < .05), or with a control MAb (35.2 +/- 4.6%, P < .01). The combination of rTPA treatment and alpha 2AP inhibition caused no consumption of fibrinogen. CONCLUSIONS: Inhibition of alpha 2AP significantly amplified the lysis of experimental pulmonary emboli by rTPA without increasing fibrinogen consumption. These results suggest that alpha 2AP may play an important role in thrombus resistance in patients with venous thromboembolism.

Identification and structure of activated-platelet protein-1, a protein with RNA-binding domain motifs that is expressed by activated platelets.

Beyond their critical role in thrombosis, platelets perform important functions in vascular remodeling, inflammation, and wound repair. Many of these functions are executed by molecules expressed by activated platelets. A novel molecule, activated-platelet protein-1 (APP-1), was identified by a monoclonal antibody against activated rabbit platelets. When platelets were stimulated by thrombin, A23187 or ADP, APP-I was expressed on the platelet surface. APP-1 was also detected in whole cell lysates of platelets, but not on the external surfaces of resting platelets. With maximal activation by thrombin, 15 900 +/- 2800 molecules APP-1 were expressed/platelet. A 2.3-kb cDNA fragment containing a partial coding sequence for APP-1 was isolated from a rabbit bone marrow library by expression cloning with the anti-APP-1 monoclonal antibody. When expressed as a recombinant fusion protein in bacteria, APP-1 bound specifically to poly(A)-Sepharose. The full-length cDNA coding for human APP-1, obtained by DNA hybridization techniques, showed 98.7% amino acid sequence identity with the rabbit protein. Northern analysis with human APP-1 identified a 3.7-kb mRNA transcript in megakaryocytic lines that express transcripts for platelet proteins. Human APP-1 has four ribonucleotide binding domains with ribonucleoprotein 1 and 2 motifs. By virtue of its ribonucleotide binding domains, APP-1 is structurally related to polyadenylate-binding protein, which regulates translation initiation and polyadenylate shortening, and to nucleolysin, a specific effector molecule found in the granules of cytotoxic T lymphocytes.

Mutation of lysines in a plasminogen binding region of streptokinase identifies residues important for generating a functional activator complex.

Through a unique but poorly understood mechanism, streptokinase (SK) interacts with human plasminogen to generate an "activator complex" that efficiently cleaves substrate plasminogen molecules. Previous studies have suggested that lysine residues in SK may play a role in the binding and function of the activator complex. To investigate this hypothesis, 10 different lysine residues in the plasminogen binding region of SK were altered to construct 8 recombinant (r) SK mutants. Only one double mutant, rSKK256,257A (replacing Lys with Ala at residues 256 and 257), showed a statistically significant reduction (63%) in binding affinity for Glu-plasminogen. This mutant also displayed a lagtime in the appearance of maximal activity, and modest impairments (2-5-fold) in kinetic parameters for amidolytic and plasminogen activator activity compared to rSK. In contrast, another mutant, rSKK332,334A, formed an activator complex with profound and nearly selective defects in the catalytic processing of substrate plasminogen molecules. When compared to rSK in kinetic assays of plasminogen activation, the rSKK332,334A mutant formed an activator complex that bound substrate plasminogens normally (normal K(m), but its ability to activate or cleave these molecules (kcat) was reduced by 34-fold. In contrast, in amidolytic assays, the kinetic parameters of rSKK332,334A showed only minor differences (< 2-fold) from rSK. Similarly, the binding affinity of this mutant to human Glu-plasminogen was indistinguishable from rSK [(2.6 +/- 0.8) x 10(9) vs (2.4 +/- 0.2) x 10(9) M-1, respectively]. In summary, these experiments have identified lysine residues in a plasminogen binding region of SK which appear to be necessary for normal high-affinity binding to plasminogen, and for the efficient catalytic processing of substrate plasminogen molecules by the activator complex.

Membrane-based cell affinity chromatography to retrieve viable cells.

A novel scheme for the separation and live recovery of one cell type from a mixture of cells using a cell affinity chromatography (CAC) system is demonstrated. An anti-murine IgG was chemically immobilized to a cellophane support via a carbonyldiimidazole (CDI) link. Murine splenocytes flowed over the support, and B-cells were allowed to attach at a shear rate of 15 s-1. Once loading was terminated, the support was washed at a shear rate of 315 s-1 to remove nonspecifically bound cells. Elution of the B-cells was initiated by the transmembrane diffusion of hydrochloric acid (pH 1), supplied to the side of the membrane opposite the cells. At the same time, a shear flow of normal saline was established on the cell side of the membrane, and cells, freed by acid, were retrieved. Results showed that, on average, 250 cells/mm2 attached to antibody immobilized on cellophane surfaces, at a shear rate of 15 s-1, and that attached cells were successfully displaced by acid supplied to the side of the membrane opposite that holding the cells. On average, at least 60% of the B-cells removed by this elution appeared viable, based on a Trypan Blue dye exclusion assay.