Isolation of an Inhibitor Selective for Collagen-stimulated Platelet Aggregation from the Soft Tick Ornithodoros moubata”

Soluble extracts from the soft tick Ornithodoros moubata were found to inhibit collagen-, ADP-, and thrombin-stimulated platelet aggregation. One inhibi- tory component was purified to homogeneity by a com-bination of gel filtration, ion-exchange, and reverse phase high pressure liquid chromatography. The purified activity, named moubatin, is a protein of molec- ular weight 17,000 and it inhibits the aggregation of washed human platelets stimulated by collagen with an ICao of -50 nM in the standard assay. At a concentration of moubatin that maximally inhibited collagen- stimulated platelet aggregation, no inhibition of aggregation initiated by other effectors, including arachi- donic acid, thrombin, ristocetin, and the calcium ionophore A23187, was observed. Moubatin also inhib- its collagen-dependent aggregation in plasma. At a higher concentration of moubatin (>1 MM) it was also possible to demonstrate an inhibitory effect on the final extent of aggregation induced by a low concentration of ADP. Although moubatin selectively inhibits plate- let activation by collagen, it has only a minimal effect on the adhesion of platelets to collagen. The amino acid sequences of peptides derived from proteolytic cleav-age of moubatin suggest that moubatin is a unique protein, consistent with its novel functional activity. and applied directly to a C1, reverse phase HPLC column which was developed with a gradient of acetonitrile in 0.1% trifluoroacetic acid. Fractions containing peptides were dried and sequenced on an AB1 gas-phase sequenator.

A wide variety of organisms obtain nourishment by sucking blood. Although the mechanism by which each carries out this process varies considerably, in most instances the predator is faced with the formidable task of maintaining the flow of blood from the tissue or blood vessel of the victim. A broad spectrum of different types of inhibitors of normal hemostasis have been found in nature, including anticoagulants, antithrombotics, and thrombolytics. Frequently these are found in the saliva of the blood sucking creature. For example, saliva of the leech Hirudo medicinalis contains hirudin (l), a potent inhibitor of thrombin, a key enzyme in the coagulation pathway as well as a potent activator of platelets. A platelet fibrinogen receptor antagonist was recently found in the saliva of another leech, Mucrobdella decoru (2). The saliva of the vampire bat contains the third type of inhibitor described above, a plasminogen activator (3). This enzyme is capable of catalyzing the dissolution of a clot.
Other less well characterized factors in the saliva of these organisms have been described which may also play a role in facilitating the flow of blood. These include nonspecific anti-* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertkement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact, $ T o whom correspondence should be addressed. Tel.: 215-652-7951; Fax: 215-652-6452. platelet activities (4, 5) and an inhibitor of collagen stimulation of platelets in the saliva of the H. medicinalis leech (6). We reported on a protein isolated from the saliva of the Huementeriu officinalis leech called LAPP (7). LAPP inhibits both the aggregation of platelets induced by collagen and also the adhesion of platelets to collagen (7, 8). The activation of platelets by other agonists is not affected by LAPP.
We recently identified and purified from Ornithodoros moubutu ticks a potent and highly specific inhibitor of blood coagulation factor Xa called TAP (9). Like hirudin, this inhibitor may serve a similar function to maintain the flow of blood by inhibiting blood coagulation. In the current study, whole body extracts from these ticks have been examined for the presence of other inhibitors of blood coagulation and platelet function. The isolation and preliminary characterization of a unique inhibitor of platelet aggregation is described.

EXPERIMENTAL PROCEDURES
Materials-Equine tendon type I collagen, arachidonic acid, ADP, and ristocetin were from Chrono-Log Corp., Havertown, PA. Thrornbin was from American Diagnostica; human fibrinogen and A23187 were from Calbiochem; Endoprotease Lys-C was purchased from Boehringer. All other reagents were from Fisher and Sigma.
Platelet Isolation and Functional Activity Assays-Platelets were isolated from healthy human volunteers free from aspirin and other drugs for at least 8 days. Blood was anticoagulated with acid citrate dextrose and the platelets washed and isolated as described previously (7). The final platelet suspension was in a modified Tyrode's buffer without Ca2+ (5 mM Hepes, 3 mM KCl, 134 mM NaCl, 0.3 mM NaH2P0,, 2 mM MgCl,, 5 mM glucose, 12 mM NaHC03, 3.5 mg/ml bovine serum albumin a t p H 7.4) adjusted to 3 X 10' platelets/ml after counting in a model ZM Coulter Counter (Coulter Electronics, Hialeah, FL). For studies in plasma, the blood was collected in 0.38% citrate and the platelet-rich plasma obtained by differential centrifugation.
Aggregation of the washed platelets was used to monitor platelet inhibitory activity in the tick soluble extracts and for each of the fractions tested throughout the purification of moubatin. The tick extract, column fraction, or aliquot of purified protein was preincubated with 0.2 or 0.25 ml of platelets a t 37 "C with 0.2 mg/ml human fibrinogen for 2 min. Collagen at 1 or 2 gglml, or another agonist, was added, and the aggregation was monitored in a Sienco or a Chronolog aggregometer. The percentage inhibition was calculated based on the final extent of aggregation of the test samples compared with the appropriate buffer control. Similar results were obtained when the rate of aggregation of the test samples compared with controls was monitored.
Analytical Techniques-SDS-PAGE' under reducing conditions was run on 20% acrylamide Phast Gels (Pharmacia Phast Gel System) and proteins were detected by staining with silver according to the protocol of the manufacturer. Protein was estimated using the bicinchoninic acid (BCA) assay (Pierce Chemical Co.) with bovine serum albumin as the standard.

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from Antibody Associates, Ltd. (Bedford, TX) and stored at -70 "C until used. A typical preparation was initiated with 400-500 whole ticks constituting about 12 g of material. The ticks were disrupted in batches of 50 with a Polytron homogenizer (Brinkmann Instruments) in 10 ml of 25 mM Hepes buffer, pH 7.0, containing 0.15 M NaCl and the protease inhibitors E-64, pepstatin, chymostatin, and leupeptin (50 pM each). The homogenate was centrifuged at 100,000 X g for 20 min, and the resulting pellets were re-extracted in 5 ml of the same buffer and recentrifuged. The combined supernatants (approximately 800 mg of protein) were tested for inhibitory activities and used for the purification of moubatin. The crude soluble extract was diluted 3-fold with water to lower the ionic strength and applied to a 25-ml Fast Q Column equilibrated in 25 mM BisTris-HC1, pH 7.0. The column was washed with the same buffer until the absorbance at 280 nm decreased to less than 0.05. Bound proteins were eluted with 0.8 M NaCl (100 ml) in the same buffer, dialyzed against water to remove the salt, and lyophilized in the presence of 0.1 mg/ml sucrose. The lyophilized material was redissolved in HzO and applied to a column of Sephadex G-50 (Superfine, 2.5 X 50 cm) equilibrated in 50 mM Hepes, pH 7.0.
The peak fractions containing platelet aggregation inhibitory activity were pooled and applied to a Pharmacia 'Mono Q anionexchange column (1 X 10 cm) equilibrated in 20 mM BisTris-HC1, pH 6.0, controlled by a fast protein liquid chromatography system. The bound proteins were eluted with a gradient of NaCl (0-0.3 M) in the same buffer. Inhibitory activity eluted in two peaks at 0.12 and 0.25 M NaCl. The active fractions in each peak were pooled separately and concentrated by lyophilization. The dried material was redissolved in 1.5 ml of HzO and applied to a gel filtration column (1.5 X 50 cm) of Sephadex (2-75 (Superfine) equilibrated in 50 mM Hepes, pH 7.0. On gel filtration each pool eluted in one peak with mass -15-20 kDa.
The final purification step consisted of fractionation of the activity pooled after gel filtration by repeated reverse phase HPLC on a Pro RPC column (0.5 X 10 cm) (Pharmacia LKB Biotechnology Inc.) controlled by a fast protein liquid chromatography system. The column was equilibrated in 0.1% (v/v) trifluoroacetic acid in water, and the bound proteins were eluted with a gradient of acetonitrile (0-50%) also in 0.1% trifluoroacetic acid. Fractions of 0.5 ml were collected, and the solvents were removed by centrifugation under vacuum in a Speed Vac concentrator. Fractions were reconstituted with -100 pl of 20 mM Hepes, pH 7.0, and assayed for platelet aggregation inhibitory activity and for purity by SDS-PAGE.
Protein Chemistry-The purified inhibitor was dissolved in a solution of 0.25 M Tris-HC1, pH 8.2, 10 mM EDTA, and 6 M guanidine hydrochloride. The protein was reduced by the addition of 50 mM dithiothreitol, and after 1 h at 37 "C, sulfhydryl groups were reacted with an excess of iodoacetamide (100 KIM) for 30 min. The alkylated protein was reisolated on a Vydac ClS reverse phase HPLC column (0.46 X 15 cm) with a gradient of acetonitrile in 0.1% trifluoroacetic acid, and the solvents were removed under vacuum. The polypeptide (20 pg) was redissolved in 100 p1 of 50 mM ammonium bicarbonate and digested with 1 pg Lys-C protease for 12 h at 37 "C. The digest was acidified and applied directly to a C1, reverse phase HPLC column which was developed with a gradient of acetonitrile in 0.1% trifluoroacetic acid. Fractions containing peptides were dried and sequenced on an AB1 gas-phase sequenator.

Characterization of Tick Extract for Inhibitory Activities-
Soluble crude extracts of 0. moubata ticks were prepared as described under "Experimental Procedures" and concentrated by lyophilization in the presence of sucrose. The dried material was redissolved in H 2 0 and fractionated by gel filtration on Sephadex G-50. The fractions were assayed for t h e presence of inhibitors of blood coagulation enzymes. Using chromogenic substrates and purified proteolytic enzymes from the coagulation cascade, inhibitors of factor Xa and thrombin were identified (Fig. 1). The inhibitor of factor Xa, TAP, has been purified to homogeneity (9), and its properties have been investigated in detail (10-13). Since platelet activation is also essential for clot formation and antiplatelet activities had been reported in hard ticks (14), we tested whether the gel filtration column fractions of the soluble extract from these Following gel filtration, the activity that inhibited collagen-stimulatedplatelet aggregation was applied to a Mono Q (1 X 10 cm) anion-exchange column equilibrated in 20 mM BisTris-HC1, pH 6.0. Bound proteins were eluted with a gradient of NaC1, and fractions were assayed for aggregation inhibitory activity as described under "Experimental Procedures." soft ticks also contained platelet aggregation inhibitors.
Aggregation dependent on collagen and thrombin was blocked by fractions from the gel filtration column. The activity that blocked thrombin-induced aggregation co-eluted with an activity that also inhibited the hydrolysis of a chromogenic substrate by thrombin. Further characterization of this activity showed that it was a thrombin inhibitor (data not shown), and it was not pursued further. The inhibitor of collagen-dependent aggregation eluted in a single peak with an apparent molecular weight of -20,000 (Fig. 1). This activity was taken for further purification.
Purification of Moubatin-The active fractions with platelet aggregation inhibitory activity from the gel filtration column were pooled and applied to an anion-exchange column (Mono Q ) , and the bound protein was eluted with a gradient of NaCl. T h i s step resolved two peaks of inhibitory activity eluting at -0.125 and 0.25 M NaCl (Fig. 2). Each of these two peaks was then purified separately.
The pools of each of the peak inhibitory fractions from the Mono Q chromatography step were lyophilized and then chromatographed on Sephadex G-75. Aggregation inhibitory activity eluted in a single peak for each sample with the same apparent molecular mass (-20 kDa) (data not shown). After gel filtration, only the activity that eluted at 0.125 M NaCl from Mono Q was purified further because of insufficient material remaining in the other sample pool. The G-75 pooled material was subjected to two additional purification steps consisting of repeated reverse phase chromatography on a Pro RPC column. Figs. 3 and 4 show that after chromatography on the second reverse phase column, the absorbance a t 214 nm and the activity co-eluted. SDS-PAGE of this material (Fig. 5) showed a single band (mass, -17 kDa) when stained with silver. We have named this inhibitor of platelet aggregation "moubatin," since it was isolated from the tick 0. moubata. Approximately 100 pg of purified protein was ob- tained from a typical preparation.
Isolation of Peptides-To obtain information on the primary structure of moubatin, the purified polypeptide was submitted to automated sequence analysis. The yield of amino acids by this procedure from -50 pmol of the purified polypeptide was less than 10% of the expected amount, suggesting that the protein was blocked at the amino terminus. Consequently, the reduced and alkylated polypeptide was digested with the protease Lys-C, which cleaves after lysyl residues. The resulting peptide fragments were then resolved by reverse phase HPLC (Fig. 6). Each of the peaks was sequenced and seven unique peptides were obtained as shown in Table I. Several of these sequences were confirmed by analysis of peptides derived from a cyanogen bromide digest of moubatin. The sequences of these peptides did not show any significant homology to collagen or any other protein in the Swiss PROT data base. Overall, the sequences correspond to 102 amino acid residues, which, based on the molecular weight of moubatin, must represent more than 50% of the total number of amino acids in the polypeptide.
Properties of Purified Moubatin-An example of the actual data obtained in experiments which examined the effect of moubatin on collagen-stimulated platelet aggregation is shown in Fig. 7A. Moubatin blocked both the rate and extent of aggregation, and at the highest concentration examined, all aggregation and shape change were completely inhibited.

Sequences of Lys-C fragments derived from moubatin
A Lys-C digest of 20 Gg of reduced and carboxymethylated mouhatin was fractionated on a CIS column. Peptides were eluted with a gradient of acetonitrile in 0.1% trifluoroacetic acid and detected by absorbance at 214 nm. The eluate was collected in an automated fraction collector and the peaks were dried down and seauenced. gregation by moubatin shown in Fig. 7B, an ICso of -50 nM was estimated. Moubatin's inhibitory activity is selective for collagen-induced platelet aggregation. The aggregation of platelets either in extent or in time course in the presence of other agonists, including ADP, arachidonic acid, thrombin, ristocetin, and the calcium ionophore A23187, was not affected (Fig. 8) by a concentration of moubatin that maximally inhibited collagen-stimulated aggregation. Moubatin also inhibited collagen-stimulated aggregation in plasma (see Table  11). However, at a concentration 10-fold above the maximum needed to inhibit collagen-stimulated aggregation, moubatin partially inhibited the final extent of aggregation induced by 2 PM ADP and to a lesser degree that induced by 20 PM ADP as shown in Table 11. Moubatin at a concentration that completely inhibited collagen-promoted aggregation had no significant effect on platelet adhesion to collagen in static adhesion assays (data not shown).

DISCUSSION
There are few specific inhibitors of collagen stimulation of platelets. We recently reported on a 16-kDa protein isolated from the leech H. officinalis, LAPP, that blocks both platelet adhesion to collagen and collagen-induced aggregation (7,8). Several other naturally occurring inhibitors of the adhesion of platelets to collagen and collagen-induced platelet aggregation have also been described. These include a 65-kDa protein from the leech H. medicinalis (6) and a 50-kDa protein from Bothrops atrox snake venom (15). However, the application of these proteins to investigate the mechanism of platelet activation by collagen and its role in blood coagulation has not yet been fully explored. Other inhibitors of collagen stimulation of platelets, including several monoclonal antibodies and collagen-derived peptides, have been shown to be useful tools to study this process, but their use has been limited by strictly defined conditions required to demonstrate their inhibitory activity (16)(17)(18)(19)(20).
The 17-kDa polypeptide that we have isolated from ticks, which is named moubatin, appears to be a selective high affinity inhibitor of collagen-dependent platelet aggregation. Using purified platelets aggregation induced by a variety of agonists is not inhibited by moubatin at a concentration that completely inhibits 1 pg/ml collagen-stimulated aggregation. Moubatin also inhibited collagen-dependent aggregation of platelets in plasma. At concentrations at least 10-fold higher than that required to block collagen-stimulated aggregation, moubatin did inhibit the second phase of platelet aggregation induced by ADP at concentrations 5 5 p~. This partial inhibitory effect was not observed with LAPP (7) and warrants further investigation. However, because this inhibitory effect is observed only at higher concentrations of moubatin, these studies cannot be carried out with native material which is available in limited quantities. We have therefore cloned and expressed this protein in large amounts, and the results of these and related investigations are presented in the accompanying paper (21).
Moubatin displays unique functional activity. Unlike the other natural product protein inhibitors of collagen stimulation of platelets, including LAPP (7), calin (6), and the B. atrox snake venom protein ( E ) , moubatin has no significant effect on the adhesion of platelets to collagen. This result has been confirmed in detailed studies utilizing recombinant moubatin as described in the accompanying report (21). Consistent with the unique functional activity of moubatin, the sequences of the 102 amino acid residues obtained from the peptides demonstrate no homology to LAPP, collagen, or to any other protein in the Swiss PROT Protein Data Bank. Also, the moubatin peptides do not contain the Arg-Gly-Asp sequence, which is the cellular recognition sequence present in extracellular matrix proteins (22) and in disintegrins (23).
Currently there are multiple cell surface receptors that have been proposed as the cellular collagen receptor. Included among these is the a&, or VLA-2, integrin complex (24). Antibodies to this protein have been shown to block platelet adhesion to collagen in static assays of adhesion (17,18). One of these antibodies, 6F1, inhibits platelet aggregation induced by collagen (17). Whether or not moubatin interacts with this cell surface integrin complex is unknown, although it is unlikely due to its lack of an inhibitory effect on adhesion of platelets to collagen. Interestingly, extracts prepared from the same ticks also contain a specific inhibitor of platelet adhesion to collagen which has no affect on collagen-induced aggrega-

TABLE I1
Effect of moubatin on platelet aggregation in plasma Platelet-rich plasma (0.25 ml) was incubated with the indicated concentration of moubatin a t 37 "C for 2 min followed by addition of agonist and aggregation was monitored as described under "Experimental Procedures." Results are expressed as the percent inhibition of the final extent of aggregation. The results shown are from one experiment representative of three separate experiments using plasma from different donors. tion.' This suggests that these two collagen-dependent processes may be mediated by a receptor with multiple affinity states or more likely by different receptors on the surface of the platelet. Why these ticks have multiple inhibitors of the platelet-collagen interaction and their importance in their feeding process are issues not yet resolved. The existence of such specific inhibitors, however, does support a role for collagen in normal hemostasis.